•PCR assays to detect key mutations of SARS-CoV-2 spike protein are described•Discrimination of Variants of Concern (VOCs) and other variants is achieved•The assays detected key mutations of ...20I/501Y.V1 and 20 J/501Y.V3 in sewage in Italy•The method allows rapid and cost-effective detection of VOCs in sewage for WBE•The described approach can be used for rapid screening in clinical samples
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.
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Human adenoviruses (HAdVs) are of major public health importance and are associated with a variety of clinical manifestations, including gastroenteritis, respiratory, ocular and urinary tract ...infections.
To study the occurrence, prevalence and diversity of HAdV species and types circulating in Italy, we conducted a large-scale molecular-epidemiological investigation, a yearlong monitoring of 22 wastewater treatment plants, covering 10 Italian regions, representative of northern, central, and southern Italy. A total of 141 raw sewage samples were collected from January to December 2013, and processed to detect and characterize by phylogenetic analysis a fragment of the hexon coding region of HAdVs.
Nested PCR results showed the presence of HAdVs in 85 out of 141 samples (60% of samples). Fifty-nine samples were characterized by conventional Sanger sequencing as belonging to four HAdV species and four types: A (type 12, 5 samples), B (type 3, 8 samples), C (type 5, 1 sample) and F (type 41, 45 samples). The remaining 26 samples could not be characterized because of uninterpretable (mixed) electropherograms suggesting the presence of multiple species and/or types.
Pools of characterized and uncharacterized PCR amplicons were further analyzed by next-generation sequencing (NGS). NGS results revealed a marked HAdV diversity with 16 additional types detected beyond the four types found by Sanger sequencing.
Overall, 19 types were identified, belonging to HAdV species A-F: types 12 and 31 (species A), type 3 (species B), types 1, 2, and 5 (species C), types 9, 17, 24, 26, 37, 38, 42, 44, 48, and 70 (species D), type 4 (species E), and types 40 and 41(species F). An untypeable HAdV was also detected, showing similar percentages of identity with more than one prototype (types 15, 30, 56, and 59).
Our findings documented the circulation of a wide variety of species and types in raw sewage, potentially able to affect other surface water environments and hence human health.
Next-generation sequencing proved to be an effective strategy for HAdV genotyping in wastewater samples. It was able to detect a wide range of “less prevalent” types unidentified by conventional Sanger sequencing, confirming that studies based on conventional technologies may grossly underestimate the existence of some, possibly less common, types.
Knowledge of the distribution of HAdV species and types would improve our understanding of waterborne HAdV-related health risks.
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•Next generation (NGS) and Sanger sequencing to detect human adenovirus in wastewater.•NGS able to detect types unidentified by conventional Sanger sequencing.•A wide variety of human adenovirus species and types detected in raw sewage.
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 × 10
8
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.
Graphic Abstract
The objective of the present study was to assess the occurrence of major waterborne enteric viruses (enterovirus, norovirus, adenovirus, rotavirus, hepatitis A and E virus) along the Tiber River in ...Italy, in areas affected by different kinds of anthropogenic pressure (agricultural, urban, industrial and pristine). Moreover, in light of the recent abundant detection of human bocavirus in urban wastewater samples in Italy, the occurrence of this virus was also assessed. Virus detection was based on nested PCR followed by sequencing, and on real‐time PCR. A correlation with anthropogenic pressure was observed. The urban and industrial areas were the most contaminated (100 and 75% of samples were positive for at least one virus respectively). The agricultural area was less contaminated, with 50% of samples positive. None of the samples collected in a pristine area were positive for viruses. The most frequently detected virus was human bocavirus, identified in 37·5% of samples, followed by norovirus and enterovirus (28% each) and adenovirus (21·6%). Rotavirus, and hepatitis A and E viruses were less common (<9%). Although Human Bocavirus is not considered a waterborne pathogen, the widespread contamination of river waters suggests that virus transmission via the water route should not be neglected.
Significance and Impact of the Study
To the best of our knowledge, this study constitutes the first attempt to assess the occurrence of enteric viruses in river waters, in areas differentially influenced by anthropogenic pressure. Enteric viruses (enterovirus, norovirus, adenovirus, rotavirus, hepatitis A and E viruses, and bocavirus) were widespread in the industrial and urban areas, and were less frequently detected in the agricultural area. Interestingly, human bocavirus was the most frequently detected virus, outnumbering even adenoviruses, known to be widespread in water environments. The widespread presence of bocavirus in surface waters suggests that a potential role of water in its transmission should not be excluded.
Significance and Impact of the Study: To the best of our knowledge, this study constitutes the first attempt to assess the occurrence of enteric viruses in river waters, in areas differentially influenced by anthropogenic pressure. Enteric viruses (enterovirus, norovirus, adenovirus, rotavirus, hepatitis A and E viruses, and bocavirus) were widespread in the industrial and urban areas, and were less frequently detected in the agricultural area. Interestingly, human bocavirus was the most frequently detected virus, outnumbering even adenoviruses, known to be widespread in water environments. The widespread presence of bocavirus in surface waters suggests that a potential role of water in its transmission should not be excluded.
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.
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.
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.
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•First detection of HEV genotype 4, subtype d, in wastewater in central Italy•Complete genome of HEV-4d was obtained by Illumina and ONT sequencing.•Importance of environmental monitoring in epidemiology and public health
Human enteric viruses are a major cause of waterborne diseases, and can be transmitted by contaminated water of all kinds, including drinking and recreational water. The objectives of the present ...study were to assess the occurrence of enteric viruses (enterovirus, norovirus, adenovirus, hepatitis A and E virus) in raw and treated wastewaters, in rivers receiving wastewater discharges, and in drinking waters. Wastewater treatment plants’ (WWTP) pathogen removal efficiencies by adenovirus quantitative real-time PCR and the presence of infectious enterovirus, by cell culture assays, in treated wastewaters and in surface waters were also evaluated. A total of 90 water samples were collected: raw and treated wastewaters (treated effluents and ultrafiltered water reused for industrial purposes), water from two rivers receiving treated discharges, and drinking water. Nested PCR assays were used for the identification of viral DNA/RNA, followed by direct amplicon sequencing. All raw sewage samples (21/21), 61.9 % of treated wastewater samples (13/21), and 25 % of ultrafiltered water samples (3/12) were contaminated with at least one viral family. Multiple virus families and genera were frequently detected. Mean positive PCRs per sample decreased significantly from raw to treated sewage and to ultrafiltered waters. Moreover, quantitative adenovirus data showed a reduction in excess of 99 % in viral genome copies following wastewater treatment. In surface waters, 78.6 % (22/28) of samples tested positive for one or more viruses by molecular methods, but enterovirus-specific infectivity assays did not reveal infectious particles in these samples. All drinking water samples tested negative for all viruses, demonstrating the effectiveness of treatment in removing viral pathogens from drinking water. Integrated strategies to manage water from all sources are crucial to ensure water quality.
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 %), and specifically in samples taken from the Atlantic Ocean and the Mediterranean Sea. The estimated concentrations of SARS-CoV-2 genome copies in the positive samples ranged from 6 to 470 per 100 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.
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•An investigation into the presence of SARS-CoV-2 RNA in distant sea and oceanic waters is presented.•The study reveals the existence of SARS-CoV-2 RNA in 16.3 % of samples, including remote oceanic waters.•The Omicron variant was detected in samples from the Atlantic Ocean and the Mediterranean Sea.•The findings emphasize the need for studies on virus circulation dynamics in marine environments.
Shellfish samples (
n
= 384) from production areas, water samples from the same areas (
n
= 39) and from nearby sewage discharge points (
n
= 29) were analyzed for hepatitis E virus (HEV) by ...real-time and nested RT-PCR. Ten shellfish samples (2.6%) and five seawater samples (12.8%) tested positive for HEV; all characterized strains were G3 and showed high degree of sequence identity. An integrated surveillance in seafood and waters is relevant to reduce the risk of shellfish-associated illnesses.
Noroviruses (NoVs) are the most frequent etiological agents of non-bacterial gastroenteritis. These viruses are transmitted through the fecal-oral route, leading to high viral loads in sewages. The ...objective of this paper was to study the environmental occurrence of the most prevalent NoV strains in different wastewater treatment plants. In addition, molecular characterization of the isolated strains was performed. Two different PCR-based methods were carried out and a novel strategy was used to verify the level of RT-PCR inhibition.
From May to September 2007, a total of 97 inflow and outflow samples were collected from five wastewater treatment plants in central Italy. We detected NoV by nested PCR in 96.9% of influent samples: 89.1% contained both genogroups; 4.7% contained only GI and 3.1% only GII. In effluents, we detected NoV in 78.8% of samples: 30.3% contained both genogroups, and 48.5% contained only GI. The major genotypes detected by sequencing analyses were GI/2, GI/5, GII/b, GII/4 and GII/6.
This work confirms the wide circulation of NoVs in Italy with a predominance of GI strains, and the widespread distribution of NoV variants in both raw and treated wastewater.
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