Molecular Epidemiology of SARS-CoV-2 in Diverse Environmental Samples Globally

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has swamped the global environment greatly in the current pandemic. Wastewater-based epidemiology (WBE) effectively forecasts the surge of COVID-19 cases in humans in a particular region. To understand the genomic characteristics/footprints and diversity of SARS-CoV-2 in the environment, we analyzed 807 SARS-CoV-2 sequences from 20 countries deposited in GISAID till 22 May 2021. The highest number of sequences (n = 638) were reported in Austria, followed by the Netherlands, China, and Bangladesh. Wastewater samples were highest (40.0%) to successfully yield the virus genome followed by a 24 h composite wastewater sample (32.6%) and sewage (18.5%). Phylogenetic analysis revealed that SARS-CoV-2 environmental strains are a close congener with the strains mostly circulating in the human population from the same region. Clade GRY (32.7%), G (29.2%), GR (25.3%), O (7.2%), GH (3.4%), GV (1.4%), S (0.5%), and L (0.4%) were found in environmental samples. Various lineages were identified in environmental samples; nevertheless, the highest percentages (49.4%) of the alpha variant (B.1.1.7) were detected in Austria, Liechtenstein, Slovenia, Czech Republic, Switzerland, Germany, and Italy. Other prevalent lineages were B.1 (18.2%), B.1.1 (9.2%), and B.1.160 (3.9%). Furthermore, a significant number of amino acid substitutions were found in environmental strains where the D614G was found in 83.8% of the sequences. However, the key mutations—N501Y (44.6%), S982A (44.4%), A570D (43.3%), T716I (40.4%), and P681H (40.1%) were also recorded in spike protein. The identification of the environmental belvedere of SARS-CoV-2 and its genetic signature is crucial to detect outbreaks, forecast pandemic harshness, and prepare with the appropriate tools to control any impending pandemic. We recommend genomic environmental surveillance to trace the emerging variants and diversity of SARS-CoV-2 viruses circulating in the community. Additionally, proper disposal and treatment of wastewater, sewage, and medical wastes are important to prevent environmental contamination.

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Key SARS-CoV-2 Mutations of Alpha, Gamma, and Eta Variants Detected in Urban Wastewaters in Italy by Long-Read Amplicon Sequencing Based on Nanopore Technology

Water ◽

10.3390/w13182503 ◽

2021 ◽

Vol 13 (18) ◽

pp. 2503

Author(s):

Giuseppina La Rosa ◽

David Brandtner ◽

Pamela Mancini ◽

Carolina Veneri ◽

Giusy Bonanno Ferraro ◽

...

Keyword(s):

Central Italy ◽

Amplicon Sequencing ◽

Wastewater Sample ◽

Global Public Health ◽

Sample Collection ◽

Increased Risk ◽

Long Read ◽

Alpha Variant ◽

Wastewater Samples ◽

Alpha Cluster

The emergence of SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs) poses an increased risk to global public health and underlines the need to prioritise monitoring and research to better respond to the COVID-19 pandemic. Wastewater monitoring can be used to monitor SARS-CoV-2 spread and to track SARS-CoV-2 variants. A long read amplicon sequencing approach based on the Oxford Nanopore technology, targeting the spike protein, was applied to detect SARS-CoV-2 variants in sewage samples collected in central Italy on April 2021. Next-generation sequencing was performed on three pooled samples. For variant identification, two approaches–clustering (unsupervised) and classification (supervised)–were implemented, resulting in the detection of two VOCs and one VOI. Key mutations of the Alpha variant (B.1.1.7) were detected in all of the pools, accounting for the vast majority of NGS reads. In two different pools, mutations of the Gamma (P.1) and Eta (B.1.525) variants were also detected, accounting for 22.4%, and 1.3% of total NGS reads of the sample, respectively. Results were in agreement with data on variant circulation in Italy at the time of wastewater sample collection. For each variant, in addition to the signature key spike mutations, other less common mutations were detected, including the amino acid substitutions S98F and E484K in the Alpha cluster (alone and combined), and S151I in the Eta cluster. Results of the present study show that the long-read sequencing nanopore technology can be successfully used to explore SARS-CoV-2 diversity in sewage samples, where multiple variants can be present, and that the approach is sensitive enough to detect variants present at low abundance in wastewater samples. In conclusion, wastewater monitoring can help one discover the spread of variants in a community and early detect the emerging of clinically relevant mutations or variants.

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Identification of Human and Animal Adenoviruses and Polyomaviruses for Determination of Sources of Fecal Contamination in the Environment

Applied and Environmental Microbiology ◽

10.1128/aem.01090-06 ◽

2006 ◽

Vol 72 (12) ◽

pp. 7886-7893 ◽

Cited By ~ 114

Author(s):

Ayalkibet Hundesa ◽

Carlos Maluquer de Motes ◽

Silvia Bofill-Mas ◽

Nestor Albinana-Gimenez ◽

Rosina Girones

Keyword(s):

River Water ◽

Water Samples ◽

Environmental Samples ◽

Fecal Contamination ◽

Source Tracking ◽

Animal Origin ◽

Urban Sewage ◽

River Water Samples ◽

Negative Results ◽

Wastewater Samples

ABSTRACT The Adenoviridae and Polyomaviridae families comprise a wide diversity of viruses which may be excreted for long periods in feces or urine. In this study, a preliminary analysis of the prevalence in the environment and the potential usefulness as source-tracking tools of human and animal adenoviruses and polyomaviruses has been developed. Molecular assays based on PCR specifically targeting human adenoviruses (HAdV), porcine adenoviruses (PAdV), bovine adenoviruses (BAdV), and bovine polyomaviruses (BPyV) were applied to environmental samples including urban sewage, slaughterhouse, and river water samples. PAdV and BPyV were detected in a very high percentage of samples potentially affected by either porcine or bovine fecal contamination, respectively. However, BAdV were detected in only one sample, showing a lower prevalence than BPyV in the wastewater samples analyzed. The 22 slaughterhouse samples with fecal contamination of animal origin showed negative results for the presence of HAdV. The river water samples analyzed were positive for the presence of both human and animal adenoviruses and polyomaviruses, indicating the existence of diverse sources of contamination. The identities of the viruses detected were confirmed by analyses of the amplified sequences. All BPyV isolates showed a 97% similarity in nucleotide sequences. This is the first time that PAdV5, BAdV6, and BPyV have been reported to occur in environmental samples. Human and porcine adenoviruses and human and bovine polyomaviruses are proposed as tools for evaluating the presence of viral contamination and for tracking the origin of fecal/urine contamination in environmental samples.

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Tracking SARS-CoV-2 in Sewage: Evidence of Changes in Virus Variant Predominance during COVID-19 Pandemic

Viruses ◽

10.3390/v12101144 ◽

2020 ◽

Vol 12 (10) ◽

pp. 1144 ◽

Cited By ~ 2

Author(s):

Javier Martin ◽

Dimitra Klapsa ◽

Thomas Wilton ◽

Maria Zambon ◽

Emma Bentley ◽

...

Keyword(s):

Viral Rna ◽

Clinical Samples ◽

Environmental Surveillance ◽

Rna Sequences ◽

Virus Variant ◽

First Case ◽

Low Levels ◽

South East England ◽

Wastewater Samples ◽

Rna Concentration

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), responsible for the ongoing coronavirus disease (COVID-19) pandemic, is frequently shed in faeces during infection, and viral RNA has recently been detected in sewage in some countries. We have investigated the presence of SARS-CoV-2 RNA in wastewater samples from South-East England between 14th January and 12th May 2020. A novel nested RT-PCR approach targeting five different regions of the viral genome improved the sensitivity of RT-qPCR assays and generated nucleotide sequences at sites with known sequence polymorphisms among SARS-CoV-2 isolates. We were able to detect co-circulating virus variants, some specifically prevalent in England, and to identify changes in viral RNA sequences with time consistent with the recently reported increasing global dominance of Spike protein G614 pandemic variant. Low levels of viral RNA were detected in a sample from 11th February, 3 days before the first case was reported in the sewage plant catchment area. SARS-CoV-2 RNA concentration increased in March and April, and a sharp reduction was observed in May, showing the effects of lockdown measures. We conclude that viral RNA sequences found in sewage closely resemble those from clinical samples and that environmental surveillance can be used to monitor SARS-CoV-2 transmission, tracing virus variants and detecting virus importations.

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Simultaneous Determination of Sildenafil and Tadalafil in Legal Drugs, Illicit/Counterfeit Drugs, and Wastewater Samples by High-Performance Liquid Chromatography

Journal of AOAC International ◽

10.5740/jaoacint.15-0320 ◽

2016 ◽

Vol 99 (4) ◽

pp. 923-928 ◽

Cited By ~ 4

Author(s):

Ali Kemal Fidan ◽

Sezgin Bakirdere

Keyword(s):

Simultaneous Determination ◽

High Performance ◽

Quantitative Measurement ◽

Correlation Coefficients ◽

Wastewater Sample ◽

Counterfeit Drugs ◽

Spiking Experiment ◽

Wastewater Samples ◽

Erectile Dysfunction Drug

Abstract A sensitive analytical method was developed for the simultaneous determination of sildenafil and tadalafil in legal drugs, illicit/counterfeit drugs, and wastewater samples. Chromatographic separation of two analytes was achieved on a C18 column with a mobile phase including 50 mM phosphate buffer at pH 6.0 and acetonitrile (35 + 65, v/v) at the flow rate of 1.0 mL/min. Analytes were separated from each other in 6 min with high resolution. LOD/LOQ values were calculated as 28/92 ng/mL for sildenafil citrate and 39/129 ng/mL for tadalafil. Calibration plots for both analytes were linear with correlation coefficients >0.9993. A validated method was successfully applied to legal and illicit erectile-dysfunction drug samples consumed in Istanbul, Turkey, and to wastewater samples. Nine different samples were analyzed for qualitative and quantitative measurement of their ingredients, and the results were compared with the values written on the labels of the drugs. The wastewater sample was also analyzed for its sildenafil and tadalafil content. To calculate the recoveries, a spiking experiment was performed and recovery rates for sildenafil and tadalafil were calculated as 101.30 ± 3.43 and 102.68 ± 1.59, respectively.

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Environmental Surveillance System To Track Wild Poliovirus Transmission

Applied and Environmental Microbiology ◽

10.1128/aem.69.5.2919-2927.2003 ◽

2003 ◽

Vol 69 (5) ◽

pp. 2919-2927 ◽

Cited By ~ 52

Author(s):

Jagadish M. Deshpande ◽

Sushmitha J. Shetty ◽

Zaeem A. Siddiqui

Keyword(s):

Environmental Samples ◽

Uttar Pradesh ◽

Immunization Coverage ◽

Paralytic Poliomyelitis ◽

Nucleotide Sequence Analysis ◽

Environmental Surveillance ◽

Poliovirus Type ◽

Wild Poliovirus ◽

Supplementary Immunization Activities ◽

Type 3

ABSTRACT Eradication of poliomyelitis from large metropolis cities in India has been difficult due to high population density and the presence of large urban slums. Three paralytic poliomyelitis cases were reported in Mumbai, India, in 1999 and 2000 in spite of high immunization coverage and good-quality supplementary immunization activities. We therefore established a systematic environmental surveillance study by weekly screening of sewage samples from three high-risk slum areas to detect the silent transmission of wild poliovirus. In 2001, from among the 137 sewage samples tested, wild poliovirus type 1 was isolated from 35 and wild poliovirus type 3 was isolated from 1. Acute flaccid paralysis (AFP) surveillance indicated one case of paralytic poliomyelitis from the city. Phylogenetic analysis with complete VP1 sequences revealed that the isolates from environmental samples belonged to four lineages of wild polioviruses recently isolated from poliomyelitis cases in Uttar Pradesh and not to those previously isolated from AFP cases in Mumbai. Wild poliovirus thus introduced caused one case of paralytic poliomyelitis. The virus was detected in environmental samples 3 months before. It was found that wild polioviruses introduced several times during the year circulated in Mumbai for a limited period before being eliminated. Environmental surveillance was found to be sensitive for the detection of wild poliovirus silent transmission. Nucleotide sequence analysis helped identify wild poliovirus reservoir areas.

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Determination of Volatile Halogenated Hydrocarbons in Wastewater by Single-Drop Microextraction Coupled to Capillary Gas Chromatography

Applied Mechanics and Materials ◽

10.4028/www.scientific.net/amm.295-298.487 ◽

2013 ◽

Vol 295-298 ◽

pp. 487-491

Author(s):

Jian Qi Sun

Keyword(s):

Gas Chromatography ◽

Capillary Gas Chromatography ◽

Halogenated Hydrocarbons ◽

Wastewater Sample ◽

Single Drop ◽

Single Drop Microextraction ◽

Relative Standard ◽

Wastewater Samples ◽

Volatile Halogenated Hydrocarbons

Single-drop microextraction (SDME) coupled to capillary gas chromatography (GC) was established for the determination of seven volatile halogenated hydrocarbons in wastewater, including chloroform (CF), carbon tetrachloride (CT), trichloroethylene (TCE), bromodichloromethane (BDCM), perchlorethylene (PCE), dibromochloromethane (DBCM) and bromoform (BF). The SDME parameters such as extractants, extracting time，stirring rate and immerging depth of needle point were studyed and optimized. The abovementioned halogenated hydrocarbons were quantified by external standardization method using GC coupled with a flame ionization detector (FID). The linear regression calibration curves, detection limits (S/N=3) and the linear ranges of the method for determining each halogenated hydrocarbons were determined, respectively. The concentrations of componds abovementioned in a real wastewater sample, the average of the recoveries obtained in the spiked wastewater samples and the corresponding relative standard deviations were determined, respectively. The results showed the proposed method several attributes, friendly enviroment, economic and highly efficient pretreatment, less time, simplicity, sensitivity, accuracy and wide linear range and so on.

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A new method for sampling African swine fever virus genome and its inactivation in environmental samples

Scientific Reports ◽

10.1038/s41598-021-00552-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Aleksandra Kosowska ◽

Jose A. Barasona ◽

Sandra Barroso-Arévalo ◽

Belén Rivera ◽

Lucas Domínguez ◽

...

Keyword(s):

Early Detection ◽

Environmental Samples ◽

Sampling Method ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Virus Genome ◽

Economic Losses ◽

Effective Vaccine ◽

Speed Up ◽

Important Basis

AbstractAfrican swine fever (ASF) is currently the most dangerous disease for the global pig industry, causing huge economic losses, due to the lack of effective vaccine or treatment. Only the early detection of ASF virus (ASFV) and proper biosecurity measures are effective to reduce the viral expansion. One of the most widely recognized risks as regards the introduction ASFV into a country is infected animals and contaminated livestock vehicles. In order to improve ASF surveillance, we have assessed the capacity for the detection and inactivation of ASFV genome by using Dry-Sponges (3 M) pre-hydrated with a new surfactant liquid. We sampled different surfaces in ASFV-contaminated facilities, including animal skins, and the results were compared to those obtained using a traditional sampling method. The surfactant liquid successfully inactivated the virus, while ASFV DNA was well preserved for the detection. This is an effective method to systematically recover ASFV DNA from different surfaces and skin, which has a key applied relevance in surveillance of vehicles transporting live animals and greatly improves animal welfare. This method provides an important basis for the detection of ASFV genome that can be assessed without the biosafety requirements of a BSL-3 laboratory at least in ASF-affected countries, which may substantially speed up the early detection of the pathogen.

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SARS-CoV-2 and Environmental Samples: A Methodological Approach to Have Consistent and Comparable Results

10.20944/preprints202010.0341.v1 ◽

2020 ◽

Author(s):

Angelo Robotto ◽

Paola Quaglino ◽

David Lembo ◽

Marcello Morello ◽

Enrico Brizio ◽

...

Keyword(s):

Environmental Samples ◽

Methodological Approach ◽

Sampling Technique ◽

Complete Information ◽

Environmental Sampling ◽

Air Samples ◽

Order Of Magnitude ◽

Wastewater Samples ◽

The Relationship ◽

Parallel Sampling

Since the beginning of coronavirus disease 2019 (COVID-19) pandemic, large attention has been focused on the relationship between SARS-CoV-2 diffusion and environment. As a matter of fact, clear evidence of the transmission of SARS-CoV-2 via respiratory aerosol would be of primary importance; at the same time, checking the presence of SARS-CoV-2 in wastewater can be extremely useful to control the diffusion of the disease. Up to now, many studies report SARS-CoV-2 concentrations in indoor/outdoor air samples or water/wastewater samples that can differ by order of magnitude. Unfortunately, complete information about the scientific approach of many studies is still missing, relating to: samplers and sampling materials performances, recovery tests, measurement uncertainty, robustness, detection and quantification limits, infectivity of captured virus, virus degradation during sampling, influence of sample pre-treatments (included freezing) on results, effects of inhibitors, sample alterations due to manipulation, validation of methods and processes, quality assurance according to ISO/IEC 17025 requirements.Based on the first experiences focused on the presence of SARS-CoV-2 in environmental samples such as air quality filters, air-liquid impingers and wastewater samples, the present study describes a coherent preliminary approach to SARS-CoV-2 environmental sampling in order to overcome the evident lack of standardization. Three aspects are highlighted here: the first solution to assure quality and consistency to environmental sampling relies on the development of recovery tests using standard materials and investigating sampling materials, sampling techniques, sampling durations, sample conservation and pre-treatments; secondly, in order to overcome the shortcomings of every single sampling technique, coupling different samplers in parallel sampling could be an efficient strategy to collect more information and make data more reliable, in particular for air samples; finally, with regards to airborne virus sampling, the results could be confirmed by simplified emission and dilution models.

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Detection and stability of SARS-CoV-2 fragments in wastewater: Impact of storage temperature

10.1101/2021.02.22.21250768 ◽

2021 ◽

Cited By ~ 1

Author(s):

Rudolf Markt ◽

Markus Mayr ◽

Evelyn Peer ◽

Andreas O. Wagner ◽

Nina Lackner ◽

...

Keyword(s):

Storage Temperature ◽

Gene Copy ◽

Wastewater Sample ◽

List Type ◽

Sample Storage ◽

Copy Numbers ◽

Wastewater Samples ◽

Gene Copy Numbers ◽

Raw Wastewater

AbstractSARS-CoV-2 wastewater epidemiology suffers from uncertainties concerning sample storage. We show the effect of storage of wastewater on the detectable SARS-CoV-2 load. Storage at 4 °C up to 9 days had no significant effect, while storage at −20 °C led to a significant reduction in gene copy numbers.HighlightsRaw wastewater samples can be stored up to 9 days at 4°C for SARS-CoV-2 wastewater surveillanceFreezing of wastewater sample dramatically decreases RT-qPCR signal of SARS-CoV-2 from wastewater.

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FIRST DETECTION OF SARS-COV-2 IN UNTREATED WASTEWATERS IN ITALY

10.1101/2020.04.25.20079830 ◽

2020 ◽

Cited By ~ 6

Author(s):

Giuseppina La Rosa ◽

Marcello Iaconelli ◽

Pamela Mancini ◽

Giusy Bonanno Ferraro ◽

Carolina Veneri ◽

...

Keyword(s):

Wastewater Treatment Plants ◽

Temporal Trends ◽

Wastewater Sample ◽

Environmental Surveillance ◽

Rna Detection ◽

Italian Case ◽

Sewer Systems ◽

Sensitive Tool ◽

Spatial And Temporal Trends ◽

Positive Results

ABSTRACTSeveral studies have demonstrated the advantages of environmental surveillance through the monitoring of sewer systems 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 occurred in volumes of 250 mL of wastewaters collected in both areas at 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 shows 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.

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