Continuous monitoring of SARS-CoV-2 RNA in urban wastewater from Porto, Portugal: sampling and analysis protocols

AbstractResearch on the emerging COVID-19 pandemic is demonstrating that wastewater infrastructures can be used as public health observatories of virus circulation in human communities. Important efforts are being organized worldwide to implement sewage-based surveillance of SARS-CoV-2 that can be used for preventive or early warning purposes, informing preparedness and response measures. However, its successful implementation requires important and iterative methodological improvements, as well as the establishment of standardized methods. The aim of this study was to develop a continuous monitoring protocol for SARS-CoV-2 in wastewater, that could be used to model virus circulation within the communities, complementing the current clinical surveillance. Specific objectives included (1) optimization and validation of a sensitive method for virus quantification; (2) monitoring the time-evolution of SARS-CoV-2 in wastewater from two wastewater treatment plants (WWTPs) in the city of Porto, Portugal. Untreated wastewater samples were collected weekly from the two WWTPs between May 2020 and March 2021, encompassing two COVID-19 incidence peaks in the region (mid-November 2020 and mid-January 2021). In the first stage of this study, we compared, optimized and selected a sampling and analysis protocol that included RNA virus concentration through centrifugation, RNA extraction from both liquid and solid fractions and quantification by reverse transcription quantitative PCR (RT-qPCR). In the second stage, we used the selected methodology to track SARS-CoV-2 in the collected wastewater over time. SARS-CoV-2 RNA was detected in 39 and 37 out of 48 liquid and solid fraction samples of untreated wastewater, respectively. The copy numbers varied throughout the study between 0 and 0.15 copies/ng RNA and a good fit was observed between the SARS-CoV-2 RNA concentration in the untreated wastewater and the COVID-19 temporal trends in the study region. In agreement with the recent literature, the results from this study support the use of wastewater-based surveillance to complement clinical testing and evaluate temporal and spatial trends of the current pandemic.

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Application of constructed wetlands for wastewater treatment in Nepal

Water Science & Technology ◽

10.2166/wst.2001.0855 ◽

2001 ◽

Vol 44 (11-12) ◽

pp. 381-386 ◽

Cited By ~ 20

Author(s):

R.R. Shrestha ◽

R. Haberl ◽

J. Laber ◽

R. Manandhar ◽

J. Mader

Keyword(s):

Wastewater Treatment ◽

Constructed Wetlands ◽

Constructed Wetland ◽

Wastewater Treatment Plants ◽

River System ◽

Present Condition ◽

Successful Implementation ◽

Urban Centers ◽

Untreated Wastewater ◽

Under Construction

Surface water pollution is one of the serious environmental problems in urban centers in Nepal due to the discharge of untreated wastewater into the river-system, turning them into open sewers. Wastewater treatment plants are almost non-existent in the country except for a few in the Kathmandu Valley and even these are not functioning well. Successful implementation of a few constructed wetland systems within the past three years has attracted attention to this promising technology. A two-staged subsurface flow constructed wetland for hospital wastewater treatment and constructed wetlands for treatment of greywater and septage is now becoming a demonstration site of constructed wetland systems in Nepal. Beside these systems, five constructed wetlands have already been designed and some are under construction for the treatment of leachate and septage in Pokhara municipality, wastewater in Kathmandu University, two hospitals and a school. This paper discusses the present condition and treatment performance of constructed wetlands that are now in operation. Furthermore, the concept of the treatment wetlands under construction is also described here. With the present experience, several recommendations are pointed out for the promotion of this technology in the developing countries.

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COVID-19 wastewater based epidemiology: long-term monitoring of 10 WWTP in France reveals the importance of the sampling context

10.1101/2021.05.06.21256751 ◽

2021 ◽

Author(s):

Adele LAZUKA ◽

Charlotte Arnal ◽

Emmanuel Soyeux ◽

Mickael Sampson ◽

Anne-Sophie Lepeuple ◽

...

Keyword(s):

Municipal Wastewater ◽

Wastewater Treatment Plants ◽

Liquid Fraction ◽

Rna Extraction ◽

Virus Concentration ◽

Municipal Wastewater Treatment ◽

Sampling Campaign ◽

Inlet Flow Rate ◽

Municipal Wastewater Treatment Plants ◽

Statistical Relationships

SARS-CoV-2 wastewater-based epidemiology (WBE) has been advanced as a relevant indicator of distribution of COVID-19 in communities, supporting classical testing and tracing epidemiological approaches. An extensive sampling campaign, including ten municipal wastewater treatment plants, has been conducted in different cities of France over a 20-weeks period, encompassing the second peak of COVID-19 outbreak in France. A well-recognised ultrafiltration - RNA extraction - RT-qPCR protocol was used and qualified, showing 5.5 +/- 0.5% recovery yield on heat-inactivated SARS-CoV-2. Importantly the whole, solid and liquid, fraction of wastewater was used for virus concentration in this study. Campaign results showed medium- to strong- correlation between SARS-CoV-2 WBE data and COVID-19 prevalence. To go further, WWTP inlet flow rate and raining statistical relationships were studied and taken into account for each WWTP in order to calculate contextualized SARS-CoV-2 loads. This metric presented improved correlation strengths with COVID-19 prevalence for WWTP particularly submitted and sensitive to rain. Such findings highlighted that SARS-CoV-2 WBE data ultimately require to be contextualised for relevant interpretation.

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Regressing SARS-CoV-2 Sewage Measurements Onto COVID-19 Burden in the Population: A Proof-of-Concept for Quantitative Environmental Surveillance

Frontiers in Public Health ◽

10.3389/fpubh.2021.561710 ◽

2022 ◽

Vol 9 ◽

Author(s):

Itay Bar-Or ◽

Karin Yaniv ◽

Marilou Shagan ◽

Eden Ozer ◽

Merav Weil ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Rna Virus ◽

Health Care Systems ◽

Rna Extraction ◽

Warning System ◽

Epidemiological Surveillance ◽

Virus Concentration ◽

Proof Of Concept ◽

Virus Rna ◽

Care Systems

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an RNA virus, a member of the coronavirus family of respiratory viruses that includes severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and the Middle East respiratory syndrome (MERS). It has had an acute and dramatic impact on health care systems, economies, and societies of affected countries during the past 8 months. Widespread testing and tracing efforts are being employed in many countries in attempts to contain and mitigate this pandemic. Recent data has indicated that fecal shedding of SARS-CoV-2 is common and that the virus RNA can be detected in wastewater. This indicates that wastewater monitoring may provide a potentially efficient tool for the epidemiological surveillance of SARS-CoV-2 infection in large populations at relevant scales. In particular, this provides important means of (i) estimating the extent of outbreaks and their spatial distributions, based primarily on in-sewer measurements, (ii) managing the early-warning system quantitatively and efficiently, and (iii) verifying disease elimination. Here we report different virus concentration methods using polyethylene glycol (PEG), alum, or filtration techniques as well as different RNA extraction methodologies, providing important insights regarding the detection of SARS-CoV-2 RNA in sewage. Virus RNA particles were detected in wastewater in several geographic locations in Israel. In addition, a correlation of virus RNA concentration to morbidity was detected in Bnei-Barak city during April 2020. This study presents a proof of concept for the use of direct raw sewage-associated virus data, during the pandemic in the country as a potential epidemiological tool.

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COVID-19 wastewater based epidemiology: long-term monitoring of 10 WWTP in France reveals the importance of the sampling context

Water Science & Technology ◽

10.2166/wst.2021.418 ◽

2021 ◽

Author(s):

A. Lazuka ◽

C. Arnal ◽

E. Soyeux ◽

M. Sampson ◽

A.-S. Lepeuple ◽

...

Keyword(s):

Municipal Wastewater ◽

Wastewater Treatment Plants ◽

Liquid Fraction ◽

Rna Extraction ◽

Virus Concentration ◽

Municipal Wastewater Treatment ◽

Sampling Campaign ◽

Inlet Flow Rate ◽

Municipal Wastewater Treatment Plants ◽

Statistical Relationships

Abstract SARS-CoV-2 wastewater-based epidemiology (WBE) has been advanced as a relevant indicator of distribution of COVID-19 in communities, supporting classical testing and tracing epidemiological approaches. An extensive sampling campaign, including ten municipal wastewater treatment plants, has been conducted in different cities of France over a 20-week period, encompassing the second peak of COVID-19 outbreak in France. A well-recognised ultrafiltration – RNA extraction – RT-qPCR protocol was used and qualified, showing 5.5 +/− 0.5% recovery yield on heat-inactivated SARS-CoV-2. Importantly the whole, solid and liquid, fraction of wastewater was used for virus concentration in this study. Campaign results showed medium- to strong- correlation between SARS-CoV-2 WBE data and COVID-19 prevalence. To go further, statistical relationships between WWTP inlet flow rate and rainfall were studied and taken into account for each WWTP in order to calculate contextualized SARS-CoV-2 loads. This metric presented improved correlation strengths with COVID-19 prevalence for WWTP particularly submitted and sensitive to rain. Such findings highlighted that SARS-CoV-2 WBE data ultimately require to be contextualized for relevant interpretation.

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Potential Use of Untreated Wastewater for Assessing COVID-19 Trends in Southern Italy

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph181910278 ◽

2021 ◽

Vol 18 (19) ◽

pp. 10278

Author(s):

Osvalda De Giglio ◽

Francesco Triggiano ◽

Francesca Apollonio ◽

Giusy Diella ◽

Fabrizio Fasano ◽

...

Keyword(s):

Disease Surveillance ◽

Southern Italy ◽

Wastewater Treatment Plants ◽

Warning System ◽

Data Interpretation ◽

World Health ◽

Virus Concentration ◽

Wastewater Sample ◽

Untreated Wastewater ◽

Health Organization

As a complement to clinical disease surveillance, the monitoring of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in wastewater can be used as an early warning system for impending epidemics. This study investigated the dynamics of SARS-CoV-2 in untreated wastewater with respect to the trend of coronavirus disease 2019 (COVID-19) prevalence in Southern Italy. A total of 210 wastewater samples were collected between May and November 2020 from 15 Apulian wastewater treatment plants (WWTP). The samples were concentrated in accordance with the standard of World Health Organization (WHO, Geneva, Switzerland) procedure for Poliovirus sewage surveillance, and molecular analysis was undertaken with real-time reverse-transcription quantitative PCR (RT-(q) PCR). Viral ribonucleic acid (RNA) was found in 12.4% (26/210) of the samples. The virus concentration in the positive samples ranged from 8.8 × 102 to 6.5 × 104 genome copies/L. The receiver operating characteristic (ROC) curve modeling showed that at least 11 cases/100,000 inhabitants would occur after a wastewater sample was found to be positive for SARS-CoV-2 (sensitivity = 80%, specificity = 80.9%). To our knowledge, this is the first study in Italy that has applied wastewater-based epidemiology to predict COVID-19 prevalence. Further studies regarding methods that include all variables (meteorological phenomena, characteristics of the WWTP, etc.) affecting this type of wastewater surveillance data would be useful to improve data interpretation.

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Online Instrumentation of Petroleum Refinery Wastewater Treatment Plants

Water Quality Research Journal ◽

10.2166/wqrj.1989.027 ◽

1989 ◽

Vol 24 (3) ◽

pp. 435-450 ◽

Cited By ~ 1

Author(s):

J.P. Stephenson

Keyword(s):

Wastewater Treatment ◽

Continuous Monitoring ◽

Wastewater Treatment Plants ◽

Petroleum Refinery ◽

Petroleum Industry ◽

Refinery Wastewater ◽

Analytical Instruments ◽

Petroleum Refinery Wastewater ◽

Sample Characterization ◽

Industry Wastewater

Abstract Several online analytical instruments are commercially available to allow continuous monitoring of petroleum industry wastewater treatment plants. Satisfactory usage of these instruments requires -special attention for sample preconditioning and sample characterization prior to selection. Specific examples of the available instruments are provided. Effective maintenance of instruments is emphasized.

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Detection of untreated sewage discharges to watercourses using machine learning

npj Clean Water ◽

10.1038/s41545-021-00108-3 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Peter Hammond ◽

Michael Suttie ◽

Vaughan T. Lewis ◽

Ashley P. Smith ◽

Andrew C. Singer

Keyword(s):

Machine Learning ◽

Wastewater Treatment ◽

Wastewater Treatment Plants ◽

Training Data ◽

Regulatory Oversight ◽

Untreated Wastewater ◽

Untreated Sewage ◽

Water Companies ◽

Analytical Approaches ◽

Sewage Discharges

AbstractMonitoring and regulating discharges of wastewater pollution in water bodies in England is the duty of the Environment Agency. Identification and reporting of pollution events from wastewater treatment plants is the duty of operators. Nevertheless, in 2018, over 400 sewage pollution incidents in England were reported by the public. We present novel pollution event reporting methodologies to identify likely untreated sewage spills from wastewater treatment plants. Daily effluent flow patterns at two wastewater treatment plants were supplemented by operator-reported incidents of untreated sewage discharges. Using machine learning, known spill events served as training data. The probability of correctly classifying a randomly selected pair of ‘spill’ and ‘no-spill’ effluent patterns was above 96%. Of 7160 days without operator-reported spills, 926 were classified as involving a ‘spill’. The analysis also suggests that both wastewater treatment plants made non-compliant discharges of untreated sewage between 2009 and 2020. This proof-of-principle use of machine learning to detect untreated wastewater discharges can help water companies identify malfunctioning treatment plants and inform agencies of unsatisfactory regulatory oversight. Real-time, open access flow and alarm data and analytical approaches will empower professional and citizen scientific scrutiny of the frequency and impact of untreated wastewater discharges, particularly those unreported by operators.

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Occurrence of SARS-CoV-2 RNA in Six Municipal Wastewater Treatment Plants at the Early Stage of COVID-19 Pandemic in The United States

Pathogens ◽

10.3390/pathogens10070798 ◽

2021 ◽

Vol 10 (7) ◽

pp. 798

Author(s):

Samendra P. Sherchan ◽

Shalina Shahin ◽

Jeenal Patel ◽

Lauren M. Ward ◽

Sarmila Tandukar ◽

...

Keyword(s):

United States ◽

Wastewater Treatment ◽

Municipal Wastewater ◽

Wastewater Treatment Plants ◽

Quantitative Polymerase Chain Reaction ◽

The United States ◽

Secondary Effluent ◽

Untreated Wastewater ◽

Treatment Processes ◽

Wastewater Samples

In this study, we investigated the occurrence of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) RNA in primary influent (n = 42), secondary effluent (n = 24) and tertiary treated effluent (n = 34) collected from six wastewater treatment plants (WWTPs A–F) in Virginia (WWTP A), Florida (WWTPs B, C, and D), and Georgia (WWTPs E and F) in the United States during April–July 2020. Of the 100 wastewater samples analyzed, eight (19%) untreated wastewater samples collected from the primary influents contained SARS-CoV-2 RNA as measured by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assays. SARS-CoV-2 RNA were detected in influent wastewater samples collected from WWTP A (Virginia), WWTPs E and F (Georgia) and WWTP D (Florida). Secondary and tertiary effluent samples were not positive for SARS-CoV-2 RNA indicating the treatment processes in these WWTPs potentially removed SARS-CoV-2 RNA during the secondary and tertiary treatment processes. However, further studies are needed to understand the log removal values (LRVs) and transmission risks of SARS-CoV-2 RNA through analyzing wastewater samples from a wider range of WWTPs.

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On-Site Pilot Testing of Hospital Wastewater Ozonation to Reduce Pharmaceutical Residues and Antibiotic-Resistant Bacteria

Antibiotics ◽

10.3390/antibiotics10060684 ◽

2021 ◽

Vol 10 (6) ◽

pp. 684

Author(s):

Sofia Svebrant ◽

Robert Spörndly ◽

Richard H. Lindberg ◽

Therese Olsen Sköldstam ◽

Jim Larsson ◽

...

Keyword(s):

Municipal Wastewater ◽

Wastewater Treatment Plants ◽

High Flow ◽

Resistant Bacteria ◽

Municipal Wastewater Treatment ◽

Antibiotic Resistant Bacteria ◽

Antibiotic Resistant ◽

Untreated Wastewater ◽

Significant Difference ◽

Hospital Sewage

Hospital sewage constitutes an important point source for antibiotics and antibiotic-resistant bacteria due to the high antibiotic use. Antibiotic resistance can develop and cause problems in sewage systems within hospitals and municipal wastewater treatment plants, thus, interventions to treat hospital sewage on-site are important. Ozonation has proven effective in treating relatively clean wastewater, but the effect on untreated wastewater is unclear. Therefore, we piloted implementation of ozonation to treat wastewater in a tertiary hospital in Uppsala, Sweden. We measured active pharmaceutical ingredients (APIs) using liquid chromatography-mass spectrometry and antibiotic-resistant Enterobacteriaceae using selective culturing pre- and post-ozonation. Comparing low (1 m3/h) and high (2 m3/h) flow, we obtained a ‘dose-dependent’ effect of API reduction (significant reduction of 12/29 APIs using low and 2/29 APIs using high flow, and a mean reduction of antibiotics of 41% using low vs. 6% using high flow, 25% vs. 6% for all APIs). There was no significant difference in the amount of antibiotic-resistant Enterobacteiaceae pre- and post-ozonation. Our results demonstrate that ozonation of untreated wastewater can reduce API content. However, due to the moderate API decrease and numerous practical challenges in the on-site setting, this specific ozonation system is not suitable to implement at full scale in our hospital.

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TaffiX® nasal powder spray forms an effective barrier against infectious new variants of SARS-CoV-2 (COVID-19)

10.21203/rs.3.rs-420365/v1 ◽

2021 ◽

Author(s):

Michal Mandelboim ◽

Ella Mendelson ◽

Yaron Drori ◽

Nofar Atari ◽

Tair Lapidot ◽

...

Keyword(s):

South African ◽

Rna Virus ◽

Rna Extraction ◽

Real Life ◽

Preclinical Study ◽

Viral Rna ◽

Pcr Analysis ◽

Effective Barrier ◽

Gel Layer

Abstract Introduction: While vaccination efforts against SARS-CoV-2 around the world are ongoing -, new high-infectious variants of the virus are being detected. The protection of the available vaccines against some of the new variants is weaker, and experts are concerned that newer as yet undescribed variants of this mutated RNA virus will eventually prove stable against the current vaccines. Additional preventive measures will therefore be needed to protect the population until effective vaccinations are widely available.TaffiX® is a personal, anti-viral nasal powder spray comprised of low pH Hypromellose that upon insufflation into the nose creates a thin gel layer covering the nasal mucosa and forming a protective mechanical barrier that prevents viruses from engaging with nasal cells- the main portal of entry for viruses. Taffix is commercially available in many countries across Europe, Asia America and Africa. In a prior preclinical study, TaffiX® was found to be effective against SARS-CoV-2 Hong Kong/VM20001061/2020 in experimental in vitro conditions. A real-life clinical survey demonstrated that TaffiX® nasal spray significantly reduced the SARS-CoV-2 infection rate post mass-gathering event in a highly endemic community.Objective: The current study aimed to test the protective effect of Taffix against new pathogenic, highly infectious SARS-CoV-2 variants in vitro: the “British” B.1.1.7 (hCoV-19/Israel/CVL-46879-ngs/2020) and the “South African” B.1.351 (hCoV-19/Israel/CVL-2557-ngs/2020) variants.Study design: A TaffiX® gel was formed on a nylon filter, using an amount equivalent to a clinical dose of Taffix . Filters were then seeded with SARS-CoV-2 B.1.1.7 (“British”) and B.1.351 (“South African”) variants. After a 10 -minute incubation at room temperature, the bottom of each filter was washed, and the resulting flow-through was collected and seeded into 24 -well plates containing Vero-E6 cells. After 5 days of incubation, a 200 µl sample from each well was taken for viral RNA extraction followed by SARS-CoV 2 RT-PCR analysis.Results: The TaffiX® gel completely blocked SARS-CoV-2 highly infectious variants B.1.1.7 and B.1.351 in vitro, reducing the titer of recoverable infectious virus as well as viral RNA by 100%.Conclusions: Under in vitro conditions, TaffiX® formed an effective protective barrier against SARS-COV-2 variants (British variant and South African Variant). These results are consistent with prior findings demonstrating the in vitro high efficacy of Taffix gel in preventing viruses from reaching cells and infecting them. These results, added to clinical real-life studies performed with Taffix , support its use as an effective barrier against new variants of SARS-CoV-2 in conjunction with other protective measures.

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