How Can Wastewater Testing Predict COVID-19 Outbreaks?

According to the CDC, over 100,000 new cases of COVID-19 are reported daily in the United States.* While diagnostic testing can provide an overview of COVID-19 infection trends, home test results are not always reported and so may not be the best solution for predicting an outbreak in advance.

In September 2020, the CDC launched the National Wastewater Surveillance System to track the presence of SARS-CoV-2 in wastewater samples collected across the country. Technology Networks spoke to Francisco Bizouarn, market development manager of the Digital Biology Group at Bio-Rad Laboratories, to find out how wastewater testing works and how it can anticipate a rise in cases of COVID-19 and the emergence of new variants.

Kate Robinson (KR): The CDC has confirmed an increased presence of COVID-19 found in wastewater samples; why is this important?

Francisco Bizouarn (FB): The CDC is relying more on monitoring wastewater for the presence of the SARS-CoV-2 virus because it indicates where the virus is present and indicates its abundance in a traceable manner. With the availability of home test kits for COVID-19, many infections are likely going unreported. Consequently, the CDC is likely only aware of a fraction of the COVID-19 cases in the US. Additionally, the virus is often detectable in wastewater days before individuals show clinical symptoms, allowing public authorities to predict new potential outbreaks and make appropriate plans to mitigate the virus’s spread and minimize its impact.

KR: What kind of response has there been to this news?

FB: Scientists agree that the US is facing a new surge in COVID-19 cases thanks to the highly contagious BA.2 variant, although recent estimates suggest that the spike will not be nearly as high as the one caused by the Omicron variant thanks to an increase in vaccination rates. Government authorities are weighing the need for protective measures against the current political climate and pandemic fatigue and are responding to this news in various ways.  Most authorities are simply telling Americans to stay vigilant. Some cities and states have begun to remove mask mandates but still encourage their use.

KR: How does wastewater surveillance work?

FB: Wastewater surveillance enables scientists to detect and quantify small concentrations of a pathogen coming from a building, community or larger region. This type of routine monitoring allows authorities to provide early warning of a disease outbreak, days or even weeks before clinical symptoms appear, making it possible for them to intervene earlier and mitigate the spread of an outbreak.

KR: What testing methods and laboratory workflows are used to quantify SARS-CoV-2 in wastewater?

FB: Clinical COVID-19 testing is usually performed using quantitative PCR (qPCR), but the more sensitive and accurate Droplet Digital^TM PCR (ddPCR^TM) technology is better suited for wastewater testing. In fact, the CDC has endorsed ddPCR as a superior technique for wastewater monitoring because it is less sensitive to inhibitors in wastewater, of which there are many. ddPCR is less sensitive to inhibitors because it quantifies that target molecule, in this case, SARS-CoV-2 RNA in an endpoint PCR analysis and is not dependent on amplification efficiency for accuracy.

Using qPCR, scientists estimate the concentration of the virus by counting the number of amplification cycles it takes for the accompanying fluorescence to reach a certain threshold. If amplification is inhibited, it will take more cycles to reach that threshold, and the qPCR result will underestimate the viral concentration in the sample. ddPCR, on the other hand, separates the sample into tens of thousands of droplets that each contain no more than a few nucleic acid strands. If a droplet contains the target viral RNA, that RNA will amplify, and a fluorescent probe will cause the droplet to light up. However, if the droplet does not contain the target sequence, the sequence will not amplify and the droplet will stay dark. The number of fluorescent (positive) and non-fluorescent (negative) droplets are counted and used to calculate the viral concentration in the original sample. Since ddPCR takes a binary measurement of whether amplification occurred (not how much amplification has taken place) in each droplet, inhibitors have less impact on accuracy.

This is especially relevant to wastewater testing. Wastewater contains various biological materials and chemicals that can disrupt a PCR reaction. Therefore, it is challenging to balance purification and sample yield: insufficient purification leaves behind residual inhibitors, and too much purification reduces the number of molecules present in the sample. Since ddPCR is less sensitive to inhibitors, it is more tolerant of less purification, meaning more molecules will remain for analysis.

KR: Should wastewater surveillance data be used alongside other data?

FB: Yes, wastewater surveillance is one part of the more extensive testing landscape for tracking the spread of SARS-CoV-2. Wastewater provides an early warning system, but authorities must pair it with clinical testing to predict and detect the future impact of different variants across different areas.

KR: How can wastewater surveillance assist in anticipating a rise in cases or the emergence of new variants? What trends have been identified from surveillance data so far?

FB: Once a new variant is identified, a new assay can be developed for wastewater testing. This will help authorities identify where new variants are emerging and predict the magnitude and direction of their spread. For example, wastewater surveillance has helped define the extent of the spread of the BA.2 variant across the US.

*Data correct as of May 25, 2022.

Francisco Bizouarn was speaking to Kate Robinson, Editorial Assistant for Technology Networks.