One of the most challenging aspects of SARS-CoV-2 is its ability to spread in asymptomatic and pre-symptomatic individuals. If there are no outward signs of COVID-19, the disease caused by the virus, how do we know who to test, isolate and contact trace? For many reasons, including cost, individual compliance, and laboratory logistics, it’s not possible to regularly test everyone. Instead, the industry is looking for ways to improve understanding of where COVID-19 has spread, with an aim to prevent outbreaks more efficiently. One of the most promising solutions that is being successfully implemented today is wastewater-based epidemiology (WBE). WBE helps public health officials detect COVID-19 by quantifying levels of viral RNA present in wastewater. This system can help to identify SARS-CoV-2 in a community before people are showing symptoms. In settings like universities, it’s a game-changer, allowing epidemiologists to intercept COVID-19 cases and to take action before a large-scale outbreak occurs. Authorities can issue restrictions and allocate resources to help manage the disease and its spread.
When a person is infected with SARS-CoV-2, the virus that causes COVID-19, it can be detected through viral RNA that has shed into the environment. Credit: NIAID.
The basics of wastewater testing
After a person is infected with SARS-CoV-2, they begin to release viral RNA through their feces into the environment for weeks. Studies have shown that the viral RNA will be present in 27 to 89 percent of people’s feces at concentrations of 0.8 to 7.5 log10 gene copies per gram for people infected with SARS-CoV-2. When shed into wastewater, the viral RNA appears to be stable for about a week It is important to note that a person will shed viral RNA even if they are asymptomatic and may continue to after they no longer test positive on a respiratory tract assay. Once the samples are taken, they can be tested using various methods. Because of the low abundance rate after dilution and the complex sample matrix, methods with high sensitivity and inhibitor tolerance, such as droplet digital PCR (ddPCR), are vital for accurate detection. The ddPCR method can either be single or multiplexed against different SARS-CoV-2 viral markers that have been established by the CDC as well as the various process controls that are used for these studies.
Depending on the sampling method and the type of ddPCR test used, wastewater testing can detect the presence of a SARS-CoV-2 infection in as few as a handful of individuals in a building, dorm or town. Early studies from a Danish hospital found that the virus could be detected in as few as two virus shedders out of 10,000. This positive test can then set off a chain of events to identify the infected individuals and determine how further spread of the infection can be prevented.
WBE testing in practice
There are many ways to approach wastewater sample collection. A series of smaller samples can be acquired over a 24-hour period (composite sampling), or a single sample can be taken during peak flow (grab sampling). With its wider timeframe, composite sampling typically provides a better overall picture than a one-time snapshot. However, grab sampling can be better for determining things that change quickly, such as bacterial levels.
Many research groups and communities are beginning to implement WBE and have seen promising results.
Epidemiologists can use wastewater treatment plants to collect wastewater from an entire community to better understand the prevalence of viruses, such as SARS-CoV-2, in that area. Credit: Chesapeake Bay Program
In September 2020, the University of Notre Dame began using WBE in select dorms to ease the burden of test administration. Kyle Bibby, PhD, associate professor at the University of Notre Dame, received a Rapid Response Research grant from the National Science Foundation to collect wastewater samples and conduct testing in the area. The University of Notre Dame administration worked with Bibby to install a continuous autosampler on the main sewage outtake. The autosampler is set up to collect wastewater every 30 minutes for 2 hours. The samples are then analyzed twice a week with ddPCR. So far, the data has been consistent with the disease burden that has been reported through other testing sources. When results showed high levels in a specific dorm, all members of the dorm were then required to get tested as a precautionary measure. By understanding how many residents actually tested positive compared to the levels recorded during WBE surveillance, Bibby was further able to confirm the potential of this type of testing and refine this method for future use.
At Colorado State University, researchers placed 17 pumps around campus. The pumps were designed to capture 85 ml samples every 15 minutes. Samples were then analyzed twice per week using ddPCR, sometimes returning results within 24 hours of collection. Because of the high sensitivity and absolute quantification of this method, the researchers can be confident about the results and understand if just one person in a building has the virus.
Researchers in Virginia also put this into practice with a 21-week study covering a region of 1.7 million people. A combination of grab and composite samples were collected weekly from nine different wastewater plants and subsequently tested with ddPCR. The researchers were able to detect the viral RNA between levels of 101 to 104 copies 100 mL?1. They used this study to help to optimize protocols, considering factors such as the assay used and the viral recovery rates for the various workflows studied.
The COVID-19 pandemic presents an opportunity for us to understand if WBE can work as a method to manage infections within communities. Some researchers are proposing a global collaboration to share data and coordinate methodologies as groups continue to learn more. Further, quantitative controls and standards can help to ensure cross-lab comparability and data defensibility.
The Sewage Analysis CORe group Europe (SCORE), the Global Water Pathogen Project and others have joined forces to launch the COVID-19 WBE Collaborative. This initiative strives to help centralize and coordinate COVID-19 WBE efforts. In addition to various communications about activities regarding WBE testing, the site will also contain protocols to help coordinate methods and an informal chat forum where members can discuss information or questions regarding WBE testing. The protocols, which include information such as sample storage or extraction techniques, will help researchers to produce comparable results and therefore produce robust and useful data sets.
As we learn more about WBE, it will be important to look at both the infrastructures in urban settings, with centralized wastewater facilities, and rural and low-income settings with decentralized wastewater systems. There may be variation between these infrastructures, so protocols for obtaining samples should take this into account. This is one reason that sharing data and protocols can help to move the field forward.
While WBE is a newer area of research, its potential for fighting the COVID-19 pandemic is revolutionary. As more use cases emerge, experts in the field must work together to share findings and protocols to maximize the value of WBE within our communities.