Brigitta Saul is commercialization manager of applied markets for Promega Corp. Saul can be reached at [email protected].
Wastewater-based epidemiology (WBE), or sewershed surveillance, has gained widespread popularity during the COVID-19 pandemic. People infected with SARS-CoV-2 shed viral particles in their feces, even before symptoms appear. The levels of SARS-CoV-2 RNA in wastewater therefore reflect the trends in infection rates in a community. Collected consistently, this data can predict major outbreaks as much as seven days earlier than clinical tests.
What is Wastewater-Based Epidemiology
Wastewater-based epidemiology is not new. It has previously been used to detect the presence of pharmaceutical or industrial waste, drug entities (including opioid abuse), viruses, and the potential emergence of super bugs.
In fact, several countries have been successful in containing Polio and Hepatitis A outbreaks using this approach.
There is an inherent bias to clinical testing that is not present in wastewater testing, however. Those coming forward for clinical testing are either unwell or have a reason to require a clinical test. That said, a single sample of wastewater provides a snapshot of an entire community, meaning its surveillance offers clear public health and cost benefits to authorities.
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Predicting COVID-19 outbreaks
A study from China[1] showed that SARS-CoV-2 RNA can be detected in human feces a few days to a week ahead of the onset of symptoms. Another study by Yale University[2] demonstrated that monitoring SARS-CoV-2 viral RNA concentration in sewage water can predict COVID-19 outbreaks seven days before individual patient testing and three days before hospital admissions.
The study concluded that WBE is a leading indicator to identify hotspots in a localized region or population. WBE can thus serve as a low cost, early warning system to identify new outbreaks, trends in current outbreaks and prevalence of infections[3].
When available, wastewater surveillance data can complement increased clinical testing and enhanced contact tracing efforts within a population, informing public health officials to either take appropriate action on containment, or relax their restrictions.
The PCR-based method commonly being used for detecting viral RNA in wastewater is very similar to what is used in clinical or research labs. However, the complexity of wastewater samples brings challenges, such as the presence of PCR inhibitors. Therefore, the viral RNA purification method is crucial for successful detection.
Current & Emerging Protocols for Detecting Viruses in Wastewater
Detection of viral material in wastewater begins by collecting samples in sewersheds, where water drains into a single point of the sewage system.
The collected wastewater samples may be pasteurized at 60°C to inactivate any live viruses. The virus is then concentrated using either a Polyethylene Glycol 8000 precipitation, centrifugal filtration or ultracentrifugation method. Extraction methods (manual, automated or high throughput) are then used to purify viral RNA from the concentrated viral material. Finally, the RNA is reverse-transcribed to cDNA and amplified via quantitative PCR using primer sets for either nucleocapsid genes (N1/N2 published by the CDC), or Envelope genes (E). The results are then analyzed and compared with ongoing levels of viral load within the areas of collection.
Innovation in this space — such as filter-based direct capture methods that can concentrate total viral nucleic acid faster than existing precipitation methods — is making WBE ever more useful in the context of a global pandemic.
Direct Capture Methods
A recent study by researchers from Promega Corp.[4] detailed how these methods overcome the shortcomings of traditional precipitation-based isolation of viral particles, which fail to capture the degraded viruses present in wastewater and are very time-consuming.
The publication demonstrates that direct capture methods enable testers to go from sample to answer in four hours and produce samples that are compatible with RT-qPCR for monitoring infection levels and next-generation sequencing (NGS) for detecting viral variants. They allow testing labs to work with the large sample volumes required for wastewater testing and remove inhibitors that can disrupt downstream testing.
In an initial concentration step, chaotropic agents were added to raw sewage allowing binding of nucleic acid from free nucleoprotein complexes, partially intact, and intact virions to a silica matrix. The eluted nucleic acid was then purified using manual or semi-automated methods. RT-qPCR enzyme mixes that would be resistant to inhibitors were formulated. Multiplexed probe-based RT-qPCR assays detecting the N1, N2 (nucleocapsid) and E (envelope) gene fragments of SARS-CoV-2 were also developed. The RT-qPCR assays contained primers and probes to detect Pepper Mild Mottle Virus (PMMoV), a fecal indicator RNA virus present in wastewater, and an exogenous control RNA to measure effects of the RT-qPCR inhibitors. Using this workflow, the researchers monitored wastewater samples from three wastewater treatment plants (WWTPs) in Dane County, Wisconsin, over the course of three months.
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The study compared the direct capture method with the widely used PEG/NaCl precipitation method for purifying SARS-CoV-2 genetic material from wastewater samples. Wastewater samples were processed using both the direct capture method (40 mL) and PEG/NaCl precipitation (120 mL) and researchers saw a 20-fold increased yield of SARS-CoV-2 RNA when using the direct capture method compared to the PEG/NaCl protocol.
The degree of concordance between the three SARS-CoV-2 targets for the three WWTPs over the sampling period (October 2020 – January 2021) was calculated using Kendall's coefficient of concordance. The researchers observed statistically significant concordance between the three SARS-CoV-2 targets for the three WWTPs.
SARS-CoV-2 RNA signals in wastewater were compared to the level of COVID-19 cases (7-day moving average) declared by the municipality. The peak of SARS-CoV-2 genetic signal observed in the wastewater was concurrent with the peak of positive SARS-CoV-2 reported in mid-November of 2020. Even with this limited data set, the potential for wastewater-based surveillance in assessing community-wide spread of the disease was confirmed.
WBE in the spotlight
Many believe that sewage surveillance is the most practical way for long-term monitoring of SARS-CoV-2 outbreaks. This approach is more cost-effective compared to large-scale diagnostic testing and can detect the virus even before symptoms have occurred, meaning public health officials can be proactive rather than reactive when controlling outbreaks.
As the pandemic continues to cause disruption to communities around the world, this useful tool is likely to prove ever more dependable as an early warning system. For routine testing personnel, novel direct capture methods offer ease-of-use and minimize the need for specialized laboratory equipment, as well as achieving consistent recovery rates and significant reduction in RT-qPCR inhibitors for reliable results.
References
- Chen Y, Chen L, Deng Q, Zhang G, Wu K, Ni L, Yang Y, Liu B, Wang W, Wei C, Yang J, Ye G, Cheng Z. The presence of SARS-CoV-2 RNA in the feces of COVID-19 patients. J Med Virol. 2020 Jul;92(7):833-840. doi: 10.1002/jmv.25825. Epub 2020 Apr 25. PMID: 32243607.
- Jordan Peccia, Alessandro Zulli, Doug E. Brackney, Nathan D. Grubaugh, Edward H. Kaplan, Arnau Casanovas-Massana, Albert I. Ko, Amyn A. Malik, Dennis Wang, Mike Wang, Daniel M. Weinberger, Saad B. Omer SARS-CoV-2 RNA concentrations in primary municipal sewage sludge as a leading indicator of COVID-19 outbreak dynamics, medRxiv 2020.05.19.20105999; doi: https://doi.org/10.1101/2020.05.19.20105999
- FQ Wu, A Xiao, JB Zhang, XQ Gu, WL Lee, K Kauffman, WP Hanage, M Matus, N Ghaeli, N Endo, C Duvallet, K Moniz, TB Erickson, PR Chai, J Thompson, EJ Alm, SARS-CoV-2 titers in wastewater are higher than expected from clinically confirmed cases, medRxiv 2020.04.05.20051540;doi: https://doi.org/10.1101/2020.04.05.20051540
- Subhanjan Mondal, Nathan Feirer, Michael Brockman, Melanie A. Preston, Sarah J. Teter, Dongping Ma, Said A. Goueli, Sameer Moorji, Brigitta Saul, James J. Cali, Promega Corporation, 5430 E Cheryl Pkwy, Fitchburg, WI 53711, United States of America, A direct capture method for purification and detection of viral nucleic acid enables epidemiological surveillance of SARS-CoV-2, Science of the Total Environment 795 (2021) 148834
