•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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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 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.
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•A real-time RT-PCR assay was designed for the rapid detection of the Omicron variant.•737 sewage samples collected throughout Italy (11 Nov – 25 Dec 2021) were tested.•The first occurrence of Omicron was on 7 December 2021, in Veneto, North Italy.•Omicron detection in sewage increased rapidly, raising from 1.0% to 65.9% in 3 weeks.•In the same period, the variant spread over the country, spreading from one Region to 17.
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.
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•332 sewage samples were collected throughout Italy in October–November 2022.•Sanger and NGS sequencing of the spike protein was used for screening of SARS-CoV-2 variants.•Prevalence of Omicron BQ.1/BQ.1.1 increased from 5 % (October) to 43 % (November).•The detection of BA.2.75 and XBB.1 was documented in November 2022.•Environmental surveillance tracked the spread of SARS-CoV-2 variants in the population.
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.
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•Enteric viruses were detected in both bathing and non-bathing water samples.•Aichivirus was the most abundant and frequently detected virus.•80% of samples with somatic coliphages were positive for enteric viruses.•Vibrio species were ubiquitous and numerous, with higher counts in August and September.
•This study aimed to assess influenza prevalence in different water environments.•Prevalence of influenza A within poultry habitats: 4.3 % to 76.4 %.•Prevalence of influenza A in migratory wild birds ...habitat: 0.4 % to 69.8 %.•Geographically: Americas 39 %, Europe 19 %, South-East Asia 2 %, Western Pacific 39 %.•Different influenza A subtypes found in 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.
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This paper addresses the issue of how water played a role in ancient conflicts, from the poisoning of water sources to flooding, to stop the advance of enemy armies. It deals with military actions ...quoted by several ancient Greek and Roman authors, who in some cases narrate these experiences first-hand. Although many abhorred such actions, they were considered tactical expedients to resort to, as cited by the war manuals of the time. The analysis starts from the ‘manual’ Strategemata of Sextus Julius Frontinus, in addition to other references left by historians and chroniclers of different periods. It continues with the evaluation of the impact of the intentional actions of water contamination described by the ancient authors, according to present toxicological and health knowledge.
A set of 11 polymorphic markers (1 cleaved amplified polymorphic sequence (CAPS), 2 sequence-characterized amplified regions (SCARs), and 8 single-nucleotide polymorphism (SNP) - derived markers) was ...obtained for olive cultivar identification by comparing DNA sequences from different accessions. Marker development was more efficient, using sequences from the database rather than cloning arbitrary DNA fragments. Analyses of the sequences of 3 genes from 11 diverse cultivars revealed an SNP frequency of 1 per 190 base pairs in exons and 1 per 149 base pairs in introns. Most mutations were silent or had little perceptible effect on the polypeptide encoded. The higher incidence of transversions (55%) suggests that methylation is not the major driving force for DNA base changes. Evidence of linkage disequilibrium in 2 pairs of markers has been detected. The set of predominantly SNP-based markers was used to genotype 65 olive samples obtained from Europe and Australia, and was able clearly to discriminate 77% of the cultivars. Samples, putatively of the same cultivar but derived from different sources, were revealed as identical, demonstrating the utility of these markers as tools for resolving nomenclature issues. Genotyping data were used for constructing a dendrogram by UPGMA cluster analysis using the simple matching similarity coefficient. Relationships between cultivars are discussed in relation to the route of olive's spread.
A specific and sensitive method based on tandem mass spectrometry with on-line high-performance liquid chromatography using atmospheric pressure chemical ionisation (LC–APCI-MS–MS) for the ...quantitation of anabolic hormone residues (17β-19-nortestosterone, 17β-testosterone and progesterone) and their major metabolites (17α-19-nortestosterone and 17α-testosterone) in bovine serum and urine is reported.
2H
217β-Testosterone was used as internal standard. The analytes were extracted from urine (following enzymatic hydrolysis) and serum samples by liquid–liquid extraction and purified by C
18 solid-phase extraction. Ionisation was performed in a heated nebulizer interface operating in the positive ion mode, where only the protonated molecule, M+H
+, was generated for each analyte. This served as precursor ion for collision-induced dissociation and two diagnostic product ions for each analyte were identified for the unambiguous hormone confirmation by selected reaction monitoring LC–MS–MS. The overall inter-day precision (relative standard deviation) ranged from 6.37 to 2.10% and from 6.25 to 2.01%, for the bovine serum and urine samples, respectively, while the inter-day accuracy (relative error) ranged from −5.90 to −3.18% and from −6.40 to −2.97%, for the bovine serum and urine samples, respectively. The limit of quantitation of the method was 0.1 ng/ml for all the hormones in bovine serum and urine. On account of its high sensitivity and specificity the method has been successfully used to confirm illegal hormone administration for regulatory purposes.
European plaice (Pleuronectes platessa) is quite frequently substituted with other flatfish, especially with Yellow fin sole (Limanda aspera), which differs not only in meat quality but also ...particularly in its origin and manipulation chain. We propose an integrated approach of laboratory and in silico mtDNA PCR-RFLP procedures generating a set of restriction patterns easily resolvable in agarose gel, able to discriminate with certainty P. platessa from other 20 flatfish species. The herein proposed procedure is an economical and valid tool in detecting mislabelled seafoods.
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