Wastewater monitoring looks set to bring extra speed and accuracy to public health measures to trace the spread of COVID-19 and, ultimately, contribute to control strategies. The Source looks at the opportunity and reviews early research from around the world.
Interest is running high around the world about the potential for testing for the SARS-CoV-2 virus in sewage, to provide information about circulation of the virus in communities.
This attraction – excitement, even – is because research has shown the approach can find the virus in sewage before the COVID-19 disease is reported. This means that sewage surveillance could be an early warning tool, for example, to screen for the virus in populations or communities. So, sewage surveillance has the potential to complement what is done in the health sector, providing information on issues that community health and public health professionals can then take steps to address. The practical implications could be vital, such as helping stem ‘second wave’ outbreaks or helping in the screening of vulnerable urban populations by testing faecal sludge of communities not served by centralised systems.
IWA has established the IWA COVID-19 Task Force as a focus for member interest and action on responding to the pandemic. One of the priority tasks identified by the group is to stimulate transfer of experience about such monitoring of the SARS-CoV-2 virus in sewage – including how to handle samples and analyse them for the virus – and how this information can be used by authorities.
Research groups around the world have worked with impressive speed to deliver the first results about monitoring the virus in sewage. As discussed below, they have shown very promising results. It is also clear from these and several later studies, however, that it is no trivial task to carry out the monitoring successfully.
Per Halkjaer Nielsen is Head of the Center for Microbial Communities, Aalborg University, Denmark, and a member of the Task Force, focusing on the surveillance area. He explains that the considerable number of studies published so far show that some standardisation and coordination are required to get everyone up to speed, and to get comparable results across studies and countries – thoughts echoed by Task Force chair Professor Joan Rose.
“As well as the task of analysing the virus using molecular methods [polymerase chain reaction (PCR)], where it can be difficult to get good, reliable quantitative results, there are issues regarding sampling, storage and handling before analysis,” says Nielsen. “Differences in these parameters make it difficult to compare results from city to city or country to country.”
Interest in the use of sewage monitoring has quickly expanded to the potential to relate the quantity of virus detected to the level of infection in a community. “The wish to translate a detected amount of virus in the sewage to the level of infections in the city population is extremely difficult, as so many parameters affect the amount analysed in the sewage,” says Nielsen.
As well as the method of analysis, these include changes in content because of transport time in the sewer, possible dilution with rainwater, temperature, and whether grab samples are used, or time or flow proportional samples. This will all strongly affect the level in the sewage, says Nielsen, adding: “There is a great need for transfer of experiences among all actors involved, and this is one of the goals of the Task Force. Besides these more analytical issues, it is also important to share experiences about the way the results should be used by authorities, so we look forward to distributing good examples.”
A review of some of the first published studies and research efforts show why there is such interest in this very fast-moving field, as well as some of the complexities.
Dr Gertjan Medema, Principal Microbiologist at KWR Water Research Institute in The Netherlands, is one of those who have been at the forefront of the efforts to progress the wastewater monitoring tool idea.
China released the genetic code of the novel coronavirus on 10 January, indicating the opportunity to use a similar type of PCR detection method to that used for the coronavirus responsible for the 2002-2004 SARS epidemic. “That is what we are using, and many others are now using, to look for this coronavirus in wastewater,” said Medema in early April.
“We started to look before the virus was reported in our country,” he said. “The virus entered our country mainly through people who went on skiing holidays in Italy and brought it back,” he added, saying that, from then, celebrations for the country’s Carnival festival, held on 23-25 February, contributed to its spread.
The initial programme of sampling was carried out in early February, early March and mid-March. This covered wastewater treatment plants serving two large cities, three serving medium-sized cities, and the country’s main airport, Schiphol. Sampling was added for the treatment plant of Tilburg, the location of the country’s index case.
Medema explained that the Dutch testing used the PCR-based method to look for four ‘signals’. These are different sequences of RNA genetic code, and the same gene fragments used in hospital laboratories to determine whether or not people have COVID-19.
The results for the first samples were negative. “Then, one week after the virus was in our country, we sampled again and found a first indication. At that time it was still inconclusive, because only one of the genetic targets produced a signal,” he said. The next samples were taken a little more than a week later, around two and a half weeks into the Dutch outbreak. “We found pretty clear signals with three of the four targets, and now we find clear signals with all four targets in all influent samples that we test for,” Medema added.
This early testing was important in demonstrating the presence of viral material at wastewater treatment plants – but the further key point is about how this connects with reports of the disease. “We have the indication it is sensitive enough to pick up virus circulation even before the health community has picked it up by notified cases,” said Medema. “We now have two examples of cities or communities where we have found the virus in sewage [and] a couple of days later, or a little over a week later, the first cases were reported.”
An important aspect of the work has been around developing the method to use, especially in terms of concentrating the virus from sewage and the isolation of RNA from the concentrate. For example, sampling was based on a 250ml, 24-hour, flow-dependent composite sample stored at 4°C during sampling. Also, Medema explained that loose RNA fragments are very unstable in sewage, so the testing process involves concentrating and purifying relatively intact viruses. He emphasised that what is detected using reverse transcriptase PCR (RT-PCR) or PCR methods is very different from what might be detected with culture-based methods, used to find viable viruses. “You find 1000-fold higher concentration differences with PCR than you find with culture methods,” he noted.
Researchers in Australia were quick to pick up on the opportunity. A team from the University of Queensland (UQ) and Australia’s national science agency CSIRO completed, in early April, what they describe in the paper presenting the first results as a proof of concept study. The progress was announced as a first step in moving to build capacity at a national scale in order to create an early warning system for tracking outbreaks.
The study looked at samples of untreated wastewater from two treatment plants in South East Queensland representing populations living in the Brisbane region. The researchers used reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) to confirm the presence of SARS-CoV-2. The study gave an estimate of 171-1090 infected people in the catchment studied, which the researchers said was in reasonable agreement with clinical observations.
“I see it as an important surveillance tool for easing restrictions,” CSIRO’s land and water science director Paul Bertsch told Reuters. “As governments ease up, they will need to keep monitoring and respond to outbreaks.”
Reuters also reported University of Queensland’s Professor Kevin Thomas as saying that it was likely that national testing would be achieved “in a matter of weeks”.
The researchers used two methods to concentrate the viral material. They also used two of the number of different RT-qPCR assays available. The positive results came from one of the assays, and the researchers said they believed the study to be the first to report detection of SARS-CoV-2 in wastewater samples with this particular assay.
In their paper, the researchers note the challenge of connecting viral RNA concentrations to the number of cases, stating: “For application of WBE [wastewater-based epidemiology] of SARS-CoV-2 infection, further systematic research is needed, covering aspects from effective sampling and preservation through to data interpretation.”
According to Reuters, the project uses an existing system under which crime agencies monitor wastewater, covering about 57% of the population, to detect the presence of illicit drugs in Australian cities.
There has also been rapid progress in the USA. In March, Massachusetts-based Biobot Analytics launched a partnership with Harvard University, Massachusetts Institute of Technology, and Brigham and Women’s Hospital for use of its technology to map and analyse the spread of the virus through wastewater.
Preliminary findings released in early April indicated that the number of COVID-19 cases was substantially greater than the fewer than 500 positive tests reported for one metropolitan region in Massachusetts.
Biobot estimated that there were in the region of 2300 to more than 115,000 infected people within the area sampled, compared with the 446 cases reported.
The researchers were reported as saying that this was the first study to estimate the number of people infected based on the levels of SARS-CoV-2 in sewage, and published in the preprint service medRxiv in early April.
The testing is based on the laboratory sending testing kits and protocols to sewage facilities, which collect 24-hour composite samples and ship them back.
Meanwhile, the University of Arizona reported at the start of April that researchers at its Water and Energy Sustainable Technology (WEST) Center were initiating a monitoring programme to test wastewater across the country to trace coronavirus prevalence in communities and help public health officials better prepare.
“We will be able to determine if the virus persists in the community even if there are no reported new cases,” said virologist Professor Charles Gerba, of the Department of Environmental Science, in a statement from the university. “To me, it’s a key to tracing the spread of a virus.”
The WEST Center is sited within the Pima County Wastewater Treatment Plant, and regularly tests for 15 different viruses in sewage and recycled waters for reuse applications. It said that it would use molecular methods and nucleic acid targets recommended by the Centers for Disease Prevention and Control (CDC) in the US to detect SARS-CoV-2 genetic markers in sewage samples collected before and after wastewater treatment.
The centre released details on its website of how municipalities could apply for sewage surveillance, including the monitoring process, sampling protocol and associated costs.
In other progress in the US, results released in the latter part of May by researchers at Yale University, in conjunction with others, reported on use of SARS-CoV-2 RNA concentrations in primary municipal sewage sludge as an indicator. This covered the COVID-19 outbreak in New Haven, Connecticut, for the period from mid-March up to the start of May.
Samples were taken each day from the treatment plant serving around 200,000 residents. The researchers report finding SARS-CoV-2 in each of the samples, and that the concentrations of virus RNA found were highly correlated with the epidemiology curve and hospital admissions. Furthermore, they also found that the RNA concentrations led the epidemiology by seven days and the hospital admissions by three days.
The researchers add that primary sludge provides a well-mixed and concentrated sample, which may be advantageous for monitoring SARS-CoV-2. On the other hand, they note that primary sludge process trains are not uniform, meaning aspects of the findings could be treatment-plant specific.
In late May, South Africa’s Water Research Commission (WRC) announced a webinar to launch a national wastewater-based epidemiology study for the surveillance of COVID-19 spread in communities.
According to WRC, epidemiological risk hotspot mapping is an approach that supports risk-informed interventions. This can combine early detection with containment and isolation. Wastewater monitoring can contribute to this, and WRC has set up a special programme to support related research. The national wastewater-based epidemiological study for the surveillance of COVID-19 spread in communities is one of the initiatives under this programme.
Announcing the programme, WRC said: “The wastewater-based epidemiology (WBE) approach could provide an effective and rapid way to predict the potential spread of novel coronavirus pneumonia (COVID-19) by picking up on biomarkers in faeces and urine from disease carriers that enter the sewer system. Rapid testing kits using paper-based devices could be used on site at wastewater treatment plants to trace sources and determine whether there are potential COVID-19 carriers in local areas. This could lead to an early warning system for COVID-19 in any second or third-wave predictions and provide preparedness for future pandemics.”
WRC used the webinar to present and introduce the South African wastewater surveillance programme and the work to be undertaken towards achieving national coverage.
Progress around the world
Many other countries are starting to analyse SARS-CoV-2 in sewage. This includes France, Germany and Switzerland, for example, as well as Brazil (see separate article). Per Nielsen explains that, as far as Denmark is concerned, a consortium of several research groups has analysed sewage from the main cities in the country, but has so far only found signals from the virus in Copenhagen, which also has the highest number of patients.
“The level of COVID-19 infections is presently very low across Denmark,” says Nielsen. “We hope to use the surveillance for early warning in case any significant virus increase happens in the populations in the major cities after the country has been opened again.”
The opportunity ahead
“It indicates that sewage surveillance could be an early warning tool, screening of the absence of the virus in populations,” said Gertjan Medema. “We think we could use sewage surveillance to complement what is now done in the health sector to survey the spread of the virus in communities.”
This includes the potential for its use in low resource settings, such as developing countries. “We see it could be an easy way to screen large populations,” he said, noting, however, the additional laboratory demands involved. Use of the approach in this context would also need to be adapted for when communities are not served by centralised treatment plants.
Medema sees the potential to assess whether the disease is increasing or decreasing, and one of the opportunities identified around the initial Dutch project was the potential for the approach to provide an early warning system for when COVID-19 may be increasing again in the country, such as during next winter. For this reason, the next step in research has been to progress a method for quantitatively assessing the presence of the virus in sewage, using RT-qPCR.
“We are now in the process of quantifying and comparing what we find in sewers to what is found in the population through health surveillance,” he said.
“There are many people around the globe that are now embarking on this. It would be super-nice if we would be able to achieve some form of global coordination collaboration on this topic.”
The work of the IWA COVID-19 Task Force is helping connect work around the world. Task Force chair Joan Rose sees a whole range of ways in which wastewater monitoring can potentially help.
She sees potential in linking trends in disease cases and infections in the community with trends in concentrations in sewage, including as a reliable predictor of trends in infections in the community where testing is low. This is very important in rapidly determining and assessing speed of spread, second waves, impacts of social distancing, and re-opening of cities.
It can help in understanding the virus circulation in the total population, as health surveillance currently sees only the tip of the iceberg. It can also help on more targeted aspects, such as identifying hotspots, providing information on racial and economic disparity, and providing early warnings (potential seasonal outbreaks) to support the health care response and vaccination.
Rose highlights broader opportunities, too, such as monitoring the uptake of vaccination, as well as creating approaches and networks that can be used to mobilise environmental monitoring if another pandemic occurs. These can also be valuable in endemic situations, using sewage surveillance to monitor circulation of pathogens and to link to the sewage surveillance of substances such as pharmaceuticals and illicit drugs, she says.
For Rose, the water sector has been playing its part as an essential service during the pandemic. Wastewater monitoring is emerging as an opportunity to strengthen that contribution: “Sewage provides a window to view the infection in the community, which has the potential to assist in benefiting society,” she says.
W Ahmed, N Angel, J Edson et al. First confirmed detection of SARS-CoV-2 in untreated wastewater in Australia: A proof of concept for the wastewater surveillance of COVID-19 in the community. Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2020.138764 (published April 14/18, 2020)
BA Kocamemi et al. First dataset on SARS-CoV-2 detection for Istanbul wastewaters in Turkey. MedRxiv, 2020.05.03.20089417 (released May 6, 2020)
G Medema et al. Presence of SARS-Coronavirus-2 in sewage. MedRxiv, 2020.03.29.20045880 (released March 30, 2020)
A Nemudryi et al. Temporal detection and phylogenetic assessment of SARS-CoV-2 in municipal wastewater. MedRxiv, 2020.04.15.20066746 (released April 20, 2020)
J Peccia et al. SARS-CoV-2 RNA concentrations in primary municipal sewage sludge as a leading indicator of COVID-19 outbreak dynamics. MedRxiv, https://doi.org/10.1101/2020.05.19.20105999 (released 22 May 2020)
W Randazzo et al. SARS-CoV-2 RNA titers in wastewater anticipated COVID-19 occurrence in a low prevalence area. MedRxiv, 2020.04.22.20075200 (released April 28, 2020)
F Wu et al. SARS-CoV-2 titers in wastewater are higher than expected from clinically confirmed cases. MedRxiv, 2020.04.05.20051540 (released April 7, 2020)
Y Wu et al. Prolonged presence of SARS-CoV-2 viral RNA in faecal samples. Lancet Gastroenterol. Hepatol., 5 (5) (2020), pp. 434-435 (published March 20, 2020)
S Wurtzer et al. Time course quantitative detection of SARS- CoV-2 in Parisian wastewaters correlates with COVID-19 confirmed cases. MedRxiv, 2020.04.12.20062679 (released May 6, 2020)
Article based on input from the IWA COVID-19 Task Force and comments made during the online discussion ‘COVID-19: A Water Professionals’ Perspective’, iwa-network.org/learn/covid-19-a-water-professionals-perspective, held 8 April 2020. Additional reporting by Keith Hayward.