Tracking the Spread of the BA.2.86 Lineage in Italy Through Wastewater Analysis.

The emergence of new SARS-CoV-2 variants poses challenges to global surveillance efforts, necessitating swift actions in their detection, evaluation, and management. Among the most recent variants, Omicron BA.2.86 and its sub-lineages have gained attention due to their potential immune evasion properties. This study describes the development of a digital PCR assay for the rapid detection of BA.2.86 and its descendant lineages, in wastewater samples. By using this assay, we analyzed wastewater samples collected in Italy from September 2023 to January 2024. Our analysis revealed the presence of BA.2.86 lineages already in October 2023 with a minimal detection rate of 2% which then rapidly increased, becoming dominant by January 2024, accounting for a prevalence of 62%. The findings emphasize the significance of wastewater-based surveillance in tracking emerging variants and underscore the efficacy of targeted digital PCR assays for environmental monitoring.

A systematic review of influenza virus in water environments across human, poultry, and wild bird habitats.

Influenza, a highly contagious acute respiratory disease, remains a major global health concern. This study aimed to comprehensively assess the prevalence of influenza virus in different aquatic environments. Using 43 articles from four databases, we thoroughly examined water matrices from wastewater treatment plants (WTPs) and other human environments, as well as poultry habitats and areas frequented by migratory wild birds. In WTP influents (10 studies), positivity rates for influenza A ranged from 0.0 % to 97.6 %. For influenza B (8 studies), most studies reported no positivity, except for three studies reporting detection in 0.8 %, 5.6 %, and 46.9 % of samples. Within poultry habitats (13 studies), the prevalence of influenza A ranged from 4.3 % to 76.4 %, while in environments frequented by migratory wild birds (11 studies), it ranged from 0.4 % to 69.8 %. Geographically, the studies were distributed as follows: 39.5 % from the Americas, 18.6 % from Europe, 2.3 % from South-East Asia and 39.5 % from the Western Pacific. Several influenza A subtypes were found in water matrices, including avian influenza (H3N6, H3N8, H4N1, H4N2, H4N6, H4N8, H5N1, H5N8, H6N2, H6N6, H7N9, H0N8, and H11N9) and seasonal human influenza (H1N1 and H3N2). The existing literature indicates a crucial requirement for more extensive future research on this topic. Specifically, it emphasizes the need for method harmonization and delves into areas deserving of in-depth research, such as water matrices pertaining to pig farming and prevalence studies in low-income countries.

Detection and full genomic sequencing of rare hepatitis E virus genotype 4d in Italian wastewater, undetected by clinical surveillance.

Hepatitis E is a liver disease caused by the hepatitis E virus (HEV), primarily transmitted through contaminated water or food. There are four different HEV genotypes in humans, with genotypes 1 and 2 being the most widespread. Genotypes 3 and 4 are found in animals and can also infect humans. Genotype 4 is prevalent in Asia, mainly in China. In Italy, only one outbreak of HEV-4 has been documented, which occurred in 2011, involving five patients. In 2013, HEV G4 was also detected in a pig farm. Since then, no further evidence of HEV genotype 4 has been found in the country. This study describes the first detection of HEV genotype 4, subtype d, in wastewater in central Italy, despite a lack of any clinical case reported in the area. By using a multiplex PCR protocol and two sequencing strategies, Illumina and ONT, the virus's complete genome was sequenced and characterized as subtype 4d. These findings shed light on the potential of environmental surveillance for infectious agents to improve our understanding of epidemiology and support public health efforts.

Exploring adenovirus in water environments: a systematic review and meta-analysis.

Adenoviruses (AdVs) have a significant impact in both medical and environmental contexts. The objective of this study was to investigate the prevalence of AdV in different water types, such as untreated and treated wastewater, surface water, groundwater, drinking water, and other water matrices. A total of 239 articles were included in this meta-analysis. Adenoviruses were detected in various waters worldwide. The overall prevalence in water was found to be 59.2%, with the highest prevalence in untreated wastewater (83.1%) and treated wastewater (75.3%), followed by "other water matrices" (53.4%), surface water (49.5%) drinking water (22.7%), and groundwater (18.5%). Most of the studies did not assess the viability of the viruses, leading to weak links between water contamination and risk. Both human and animal AdV were found in water environments. The findings suggest that water, including drinking water, could be a significant route of AdV transmission in both developed and developing economies.

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.

Hepatitis E Virus in Water Environments: A Systematic Review and Meta-analysis.

Hepatitis E virus (HEV) is responsible for acute hepatitis in humans, through foodborne, zoonotic, and waterborne transmission routes. This study aimed to assess the prevalence of HEV in water matrices. Six categories were defined: untreated and treated wastewater, surface water (river, lake, and seawater), drinking water, groundwater, and other water environments (irrigation water, grey water, reservoir water, flood water, and effluent of pig slaughterhouse). We searched PubMed, Web of Science, Global Index Medicus, and Excerpta Medica Database. Study selection and data extraction were performed by at least two independent investigators. Heterogeneity (I2) was assessed using the χ2 test on the Cochran Q statistic and H parameter. Sources of heterogeneity were explored by subgroup analysis. This study is registered with PROSPERO, number CRD42021289116. We included 87 prevalence studies from 58 papers, 66.4% of which performed in Europe. The overall prevalence of HEV in water was 9.8% (95% CI 6.4-13.7). The prevalence was higher in untreated wastewater (15.1%) and lower in treated wastewater (3.8%) and in drinking water (4.7%). In surface water, prevalence was 7.4%, and in groundwater, the percentage of positive samples, from only one study available, was 8.3%. Overall, only 36.8% of the studies reported the genotype of HEV, with genotype 3 (HEV-3) prevalent (168 samples), followed by HEV-1 (148 sample), and HEV-4 (2 samples). High-income countries were the most represented with 59/87 studies (67.8%), while only 3/87 (3.5%) of the studies were performed in low-income countries. The overall prevalence obtained of this study was generally higher in industrialized countries. Risk of bias was low in 14.9% of the studies and moderate in 85.1%. The results of this review showed the occurrence of HEV in different waters environments also in industrialized countries with sanitation and safe water supplies. While HEV transmission to humans through water has been widely demonstrated in developing countries, it is an issue still pending in industrialized countries. Better knowledge on the source of pollution, occurrence, survival in water, and removal by water treatment is needed to unravel this transmission path.

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance.

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5-11 December, to 17.5% (25/143 samples) in the week 12-18, to 65.9% (89/135 samples) in the week 19-25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool.

A State-of-the-Art Scoping Review on SARS-CoV-2 in Sewage Focusing on the Potential of Wastewater Surveillance for the Monitoring of the COVID-19 Pandemic.

The outbreak of coronavirus infectious disease-2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has rapidly spread throughout the world. Several studies have shown that detecting SARS-CoV-2 in untreated wastewater can be a useful tool to identify new outbreaks, establish outbreak trends, and assess the prevalence of infections. On 06 May 2021, over a year into the pandemic, we conducted a scoping review aiming to summarize research data on SARS-CoV-2 in sewage. Papers dealing with raw sewage collected at wastewater treatment plants, sewer networks, septic tanks, and sludge treatment facilities were included in this review. We also reviewed studies on sewage collected in community settings such as private or municipal hospitals, healthcare facilities, nursing homes, dormitories, campuses, airports, aircraft, and cruise ships. The literature search was conducted using the electronic databases PubMed, EMBASE, and Web Science Core Collection. This comprehensive research yielded 1090 results, 66 of which met the inclusion criteria and are discussed in this review. Studies from 26 countries worldwide have investigated the occurrence of SARS-CoV-2 in sewage of different origin. The percentage of positive samples in sewage ranged from 11.6 to 100%, with viral concentrations ranging from ˂LOD to 4.6 × 108 genome copies/L. This review outlines the evidence currently available on wastewater surveillance: (i) as an early warning system capable of predicting COVID-19 outbreaks days or weeks before clinical cases; (ii) as a tool capable of establishing trends in current outbreaks; (iii) estimating the prevalence of infections; and (iv) studying SARS-CoV-2 genetic diversity. In conclusion, as a cost-effective, rapid, and reliable source of information on the spread of SARS-CoV-2 and its variants in the population, wastewater surveillance can enhance genomic and epidemiological surveillance with independent and complementary data to inform public health decision-making during the ongoing pandemic.

An innovative approach for the non-invasive surveillance of communities and early detection of SARS-CoV-2 via solid waste analysis.

The diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection requires the detection of viral RNA by reverse transcription-polymerase chain reaction (RT-qPCR) performed mainly using nasopharyngeal swabs. However, this procedure requires separate analysis per each individual, performed in advanced centralized laboratory facilities with specialized medical personnel. In this study, an alternative approach termed "solid waste-based surveillance (SWBS)" was explored, in order to investigate SARS-CoV-2 infection in small communities through the indirect sampling of saliva left on waste. Sampling was performed at 20 different sites in Italy during the second peak of COVID-19. Three swabs were positive for SARS-CoV-2 using a published RT-qPCR protocol targeting the non-structural protein 14 region, and the viral load ranged 4.8 × 103-4.0 × 106 genome copies/swab. Amino acid substitutions already reported in SARS-CoV-2 sequences circulating in Italy (A222V and P521S) were detected in two positive samples. These findings confirmed the effectiveness of SWBS for non-invasive and dynamic SARS-CoV-2 surveillance.

Rapid screening for SARS-CoV-2 variants of concern in clinical and environmental samples using nested RT-PCR assays targeting key mutations of the spike protein.

New SARS-CoV-2 mutations are constantly emerging, raising concerns of increased transmissibility, virulence or escape from host immune response. We describe a nested RT-PCR assay (~1500 bps) to detect multiple nucleotide changes resulting in key spike protein mutations distinctive of the major known circulating SARS-CoV-2 variants, including the three Variants of Concern (VOCs) 20I/501Y.V1 (United Kingdom), 20H/501Y.V2 (South Africa), and 20 J/501Y.V3 (Brazil), as well as the 20E.EU1 variant (Spain), the CAL.20C recently identified in California, and the mink-associated variant (GR, lineage B.1.1.298). Prior to application to field samples, the discriminatory potential of this PCR assay was explored using GISAID and Nextclade. To extend variant detection to challenging matrices such as sewage, where the amplification of long fragments is problematic, two short nested RT-PCR assays (~300 bps) were also designed, targeting portions of the region spanned by the long nested assay. The three newly-designed assays were then tested on field samples, including 31 clinical samples (7 fully-sequenced swab samples, and 24 uncharacterized ones) and 34 urban wastewater samples, some of which collected in areas where circulation of VOCs had been reported. The long assay successfully amplified 29 of the 31 swabs (93%), allowing the correct identification of variants 20I/501Y.V1 and 20E.EU1 present in the panel of previously characterized samples. The Spanish variant was detected in 14/24 of the uncharacterized samples as well. The sequences obtained using the short assays were consistent with those obtained with the long assay. Mutations characteristic of VOCs (UK and Brazilian variant) and of other variant (Spanish) were detected in sewage samples. To our knowledge, this is the first evidence of the presence of sequences harboring key mutations of 20I/501Y.V1 and 20 J/501Y.V3 in urban wastewaters, highlighting the potential contribution of wastewater surveillance to explore SARS-CoV-2 diversity. The developed nested RT-PCR assays can be used as an initial rapid screening test to select clinical samples containing mutations of interest. This can speed up diagnosis and optimize resources since it allows full genome sequencing to be done only on clinically relevant specimens. The assays can be also employed for a rapid and cost-effective detection of VOCs or other variants in sewage for the purposes of wastewater-based epidemiology. The approach proposed here can be used to better understand SARS-CoV-2 variant diversity, geographic distribution and impact worldwide.

Pepper Mild Mottle Virus as Indicator of Pollution: Assessment of Prevalence and Concentration in Different Water Environments in Italy.

Pepper mild mottle virus (PMMoV), a plant pathogenic virus belonging to the family Virgoviridae, has been proposed as a potential viral indicator for human faecal pollution in aquatic environments. The present study investigated the occurrence, amount and diversity of PMMoV in water environments in Italy. A total of 254 water samples, collected between 2017 and 2019 from different types of water, were analysed. In detail, 92 raw sewage, 32 treated sewage, 16 river samples, 9 estuarine waters, 20 bathing waters, 67 groundwater samples and 18 drinking waters were tested. PMMoV was detected in 79% and 75% of untreated and treated sewage samples, respectively, 75% of river samples, 67% and 25% of estuarine and bathing waters and 13% of groundwater samples. No positive was detected in drinking water. The geometric mean of viral concentrations (genome copies/L) was ranked as follows: raw sewage (2.2 × 106) > treated sewage (2.9 × 105) > river waters (6.1 × 102) > estuarine waters (4.8 × 102) > bathing waters (8.5 × 101) > groundwater (5.9 × 101). A statistically significant variation of viral loads could be observed between raw and treated sewage and between these and all the other water matrices. PMMoV occurrence and viral loads did not display seasonal variation in raw sewage nor correlation with faecal indicator bacteria in marine waters and groundwater. This study represents the first report on the occurrence and quantification PMMoV in different water environments in Italy. Further studies are required to evaluate the suitability of PMMoV as a viral indicator for human faecal pollution and for viral pathogens in waters.

Evidence of Saffold virus circulation in Italy provided through environmental surveillance.

Saffold virus (SAFV) is an emerging human cardiovirus associated with respiratory and gastrointestinal infection, and, more recently, to symptoms related to the endocrine, cardiovascular, and neurological systems. Information about SAFV circulation in Italy is scarce. In order to provide insights into the epidemiology of SAFV in Italy, 141 raw sewage samples collected throughout Italy were tested using broad-range nested RT-PCR primers targeting the 5'-NC region. Seven samples (5·0%) were confirmed as SAFV in samples collected in North, Centre and Southern Italy. Typing was attempted through amplification of the VP1 coding region, using both published and newly designed primers, and one sample was characterized as SAFV-2. SIGNIFICANCE AND IMPACT OF THE STUDY: Prevalence, genetic diversity and geographic distribution of SAFV in Italy is currently unknown. This study represents the first detection of SAFV in sewage samples in Italy, suggesting that it is circulating in the population despite lack of clinical reporting. Whether the virus is associated with asymptomatic cases or with undetected gastroenteritis or respiratory illness is unknown. Further studies are needed to investigate on the occurrence and persistence of SAFV in water environments and its waterborne transmission potential.

Enteric viruses, somatic coliphages and Vibrio species in marine bathing and non-bathing waters in Italy.

Microbial safety of recreational waters is a significant public health issue. In this study we assessed the occurrence and quantity of enteric viruses in bathing and non-bathing waters in Italy, in parallel with microbial faecal indicators, somatic coliphages and Vibrio spp. Enteric viruses (aichivirus, norovirus and enterovirus) were detected in 55% of bathing water samples, including samples with bacterial indicator concentrations compliant with the European bathing water Directive. Aichivirus was the most frequent and abundant virus. Adenovirus was detected only in non-bathing waters. Somatic coliphages were identified in 50% bathing water samples, 80% of which showed simultaneous presence of viruses. Vibrio species were ubiquitous, with 9 species identified, including potential pathogens (V. cholerae, V. parahaemoylticus and V. vulnificus). This is the first study showing the occurrence and high concentration of Aichivirus in bathing waters and provides original information, useful in view of a future revision of the European Directive.

Evidence for swine and human papillomavirus in pig slurry in Italy.

AIMS: The diversity and the geographical distribution of swine papillomaviruses (PVs) are virtually unknown. The occurrence and the diversity of swine PV were therefore investigated in pig slurry collected in Italy, to contribute towards filling this gap in knowledge. METHODS AND RESULTS: Twenty-two slurry samples underwent analysis by nested PCR and DNA sequencing using published and newly designed specific primer pairs for Sus scrofa papillomavirus (SsPV) type 1 and 2 (SsPV1 and 2), along with degenerate PV-specific primers targeting the major coat protein L1 and the helicase protein E1. Overall, three samples (13·6%) were positive for SsPV1 by specific primers, and nucleotide (nt) sequences showed 99-100% nt identity with SsPV1 variant a (EF395818), while SsPV2 was not found in any sample. Using generic primers, eight samples (36·4%) were tested positive for human papillomavirus (HPV), and were characterized as follows: ß1-HPV8, ß1-HPV14, ß1-HPV206, ß2-HPV113, ß2-HPV120 and γ1-HPV173. Moreover, one unclassified γ-type was detected. CONCLUSIONS: Both swine and human PVs were detected in pig slurry in this study. The unexpected presence of HPV in pig waste could be explained as the result of an improper use of the sewage collection pits and/or with improper procedures of the operators. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports the first detection of SsPV1 in Italy, along with the first detection of HPVs in pig slurry samples in Italy, and expands our knowledge about PV diversity and geographic distribution.

Prenatal exposure to methylmercury during late gestation affects cerebral opiatergic system in rat offspring.

Pregnant female rats were orally administered a single dose (8 mg/kg) of methylmercury chloride (MMC) on Day 15 of gestation. The binding characteristics of opioid receptors were studied in the brain of developing rats at different stages of age. An increased density of opioid receptors was found in whole brain of MMC-exposed rats at 21 days (delta receptors) and 60 days (mu and delta receptors) of age, in comparison with matched controls. An enhanced response to morphine administration was detected in MMC-exposed rat offspring at Day 60 of postnatal life, which, however, was not apparently due to an impaired liver metabolization or renal excretion. Hence, it is reasonable to surmise a possible correlation between receptor up-regulation and increased response to pharmacological challenge. These data seem to indicate that neurochemical alterations produced in the rat developing organism by prenatal exposure to methylmercury involve the opiatergic system which undergoes a supersensitivity phenomenon. This effect, which is not detectable in the first postnatal period, shows a delayed onset, being detectable only at the adult stage. These findings seem to indicate that pre- and postnatal methylmercury exposure induces latent neurochemical and behavioral alterations which could last even after the clearance of the metal from the brain.

Methyl mercury during late gestation affects temporarily the development of cortical muscarinic receptors in rat offspring.

Pregnant Sprague-Dawley rats were treated by gavage with a single dose of 8 mg/kg of methyl mercury on gestational day 15. Offspring of control and treated rats were killed at 14, 21 and 60 days of age. The binding characteristics of muscarinic receptors labelled in cortical membrane preparation by 3H-L-quinuclidinyl benzilate were studied together with the assessment of mercury level in the same brain area. Furthermore, the performance in passive avoidance tasks was evaluated in 8 weeks old rats. Perinatal exposure to methyl mercury significantly reduced the maximum number of muscarinic receptors (Bmax) in the brain of 14 (53%) and 21 day old rats (21%), while this change was no more present in 60 day old rats. This phenomenon seems to be strictly related to the presence of mercury in the cortex since it disappeared with the normalization of mercury levels in the brain. Despite the recovery of muscarinic receptor densities in methyl mercury exposed rats at 8 weeks of age, the avoidance latency was reduced in passive avoidance test as an indication of learning and memory deficits in these animals. Results from this study indicate that prenatal methyl mercury exposure induces latent cognitive dysfunction which does not seem to be related to transient muscarinic receptor alteration found in the early period of postnatal life.

Synthesis and antiinflammatory activity of 2,6-bis(1,1-dimethylethyl)phenol derivatives.

The influence of 11 newly synthesized 2,6-bis(1,1-dimethylethyl)phenol derivatives substituted in the 4 position as measured on the carrageenan paw edema assay in Sprague-Dawley rats, was studied using indomethacin as a reference drug. Furthermore we studied the possible interference of few of these compounds on the calcium binding sites by using the specific ligand [3H]-PN 200-110 in "in vitro" experiments. As far as regard the antiinflammatory activity only the compounds 2b, 2j and 2k, dosed at 20 mg/Kg/os, exerted an inhibitory effect on paw edema which was practically equal, after 6 h, to that of indomethacin (approximately 30%) dosed at 2.5 mg/Kg. The compound 2k, however, showed, in comparison with indomethacin and the other new tested compounds, a longer lasting effect, reaching, after 8 h, a 56.7% inhibition of the edema. Finally the above mentioned compounds, when tested alone or in combination with nitrendipine, did not exert any displacing activity on [3H]-PN 200-110 binding to synaptosomal membranes. It is noteworthy however that compound 2e, which incidentally was inactive as antiinflammatory agent, showed a negative allosteric modulatory activity on the ability of nitrendipine to displace [3H]-PN 200-110 binding.