SARS-CoV-2 RNA detection in wastewater can be a powerful surveillance tool for future local outbreaks
When dealing with a global pandemic such as COVID-19, every day counts. To better respond to a local surge in infections, early detection is of vital importance. However, days often go between the increase in new infections and detection by a clinical test. Recently, research has shown that SARS-CoV-2 spread can be detected more quickly in wastewater. In fact, the examination of wastewater enables faster identification of sources of infection by pool-testing entire communities long before clinical swab testing.How does SARS-CoV-2 end up in wastewater?
People who have become infected excrete the SARS-CoV-2 virus after two to three days. Once in the sewage, viruses can be damaged by bacteria, heat and cold in the wastewater. However, their genetic material remains intact for days. This allows researchers to not only detect the virus in waste but once found; it can be genetically sequenced to figure out how much it has mutated. Research has shown that the virus can be detected in the human stool within hours after infection, independent of the patient showing no, mild or strong COVID-19 symptoms.
Consequently, the presence of the virus has been detected in some wastewater treatment facilities before the virus spread in the population, and medical symptoms were reported. The time between the appearance of the virus in untreated wastewater and an increase in the number of symptomatic patients oscillates between a few days to a few weeks, making wastewater testing an ideal early warning system.
Finding SARS-CoV-2 RNA in wastewater
In the spring of 2020, China reported detecting the SARS-CoV-2 virus in the feces of infected people. Shortly after, a collaboration between the University of Queensland and the Commonwealth Scientific and Industrial Research Organization (CSIRO), Australia’s national science agency, initiated work exploring the possibility of testing for SARS-CoV-2 in raw sewage.
This was a seemingly complex research question. No one knew at the time what happened after the waste was flushed away to a wastewater treatment plant and mixed with the outflow from various other sources. Were the virus’ genetic remnants still detectable? Back then, testing methods for COVID-19 were still in development, and there was a pressing need to find new ways to track and trace the scope and scale of the pandemic.
Dr. Warish Ahmed, a senior research scientist at CSIRO Land & Water, recalls when the samples collected at treatment plants handling wastewater from Brisbane arrived at his laboratory, and a highly sensitive analytical method developed by his team revealed the first confirmed evidence of SARS-CoV-2 RNA in wastewater in Australia.
Using a simple model, they roughly estimated that 171–1090 people out of the approximately 600,000 people covered in the sampling area were potentially infected. “The results were proof this method could potentially be used to identify hotspots and infected people,” says Ahmed. This proof-of-concept became the basis for what is now a cost-effective way to test entire communities for COVID-19 infections globally.
Wastewater-based epidemiology (WBE) as a tool for COVID-19 surveillance in Australia
What started as a collaborative pilot study between CSIRO, the University of Queensland and Queensland Health to incorporate WBE into public health has become a routine monitoring program, analyzing more than 1900 samples to date.With the increasing number of samples, an automated system for sample preparation became a must-have. The team uses two QIAcube Connect for automated RNA extraction from concentrated sewage samples. Suzanne Metcalfe, a research projects officer who collaborates with Ahmed on several projects, says, "the QIAcube platform is robust and consistent in its results. It is easy to use and increases productivity in the lab."
Wastewater samples are notoriously difficult to process due to high levels of PCR inhibitors, including polysaccharides and other secondary metabolites. The QIAcube Connect, in combination with the RNeasy PowerMicrobiome Kit based on the patented Inhibitor Removal Technology (IRT), provided pure and inhibitor-free RNA, significantly reducing the chance of contamination and downstream inhibition.
WBE took flight
The value of WBE was soon being realized for smaller-scale monitoring: sampling wastewater from nursing homes, retirement villages, cruise ships and even planes with repatriating Australians. “We’ve been able to report back to Qantas on which planes have come in with positive samples,” says Metcalfe.Their results have indicated about 90% accuracy of detecting COVID-19 by analyzing wastewater compared to clinical swab testing.
Future of wastewater testing for SARS-CoV-2
Despite vaccines becoming more available, the appearance of new variants still indicates a need for public health agencies to monitor COVID-19 circulation on a mass – and affordable – scale. WBE is creating waves around the world. Many European countries are implementing wastewater COVID-19 detection programs, with The Netherlands monitoring the wastewater of all treatment plants in the country. And last year, the US launched a nationwide wastewater surveillance program for public health departments.
Based on European Union estimates, upscaling costs for national rollouts are estimated to amount to 1–3 Mio € per Member State, depending on the number of water treatment plants included in the surveillance program.
Altogether, this shows the many advantages of wastewater testing including lower costs and the possibility of screening thousands of people at once. “WBE is cheaper than individual testing, which costs about $75 per person and gives just a snapshot of a single person at a particular time. If you monitor wastewater, you can potentially screen 10,000 people, 100,000 people, by analyzing one sample,” Ahmed says.
The need for absolute quantification
While vaccines will reduce the number of COVID-19 infections and lower the viral load of infected people, making it hard to detect the virus at such low concentrations, incorporating new sensitive detection methods, such as digital PCR, might increase sensitivity five- to ten-fold. Digital PCR is a highly precise approach to sensitive and reproducible nucleic acid detection and quantification. It is proving its superiority in many of the applications that currently use quantitative PCR, among other things, testing for SARS-CoV-2 RNA virus in wastewater. The QIAcuity nanoplate digital PCR system partitions the sample into 26,000 individual reactions, increasing the chances of identifying a single positive. More on this story can be found here.
From Australia to Europe, scientists are discovering the value of digital PCR in population-wide disease monitoring. Digital PCR also proved useful in SARS-CoV-2 virus detection in a French oyster farm. As filtrating animals, oysters are very susceptible to the quality of the water where they live and, therefore, the impact of wastewater overflow. The French startup I.A.G.E., Montpellier, took advantage of the QIAcuity dPCR solution because of its throughput, reliability, simplicity of use and affordable price, revealing not only the presence of the virus but also the first proof that the UK variant had reached the country. It has since allowed them to perform global and massive monitoring of the infection levels in a population within a city, and even smaller communities, such as residential settings for elderly people. You can read the full story here.
To find out more about how QIAGEN is supporting COVID-19 wastewater testing applications, visit the COVID-19 hub.