In 2019, the use of wastewater-based epidemiology on a large scale was closer to a pipe dream than reality. Five years and one global pandemic later, wastewater-based epidemiology (WBE) is heralded for the data collection potential it provides for a vast array of pathogens.
Tracing the cellular rudiments of disease-causing organisms and viruses excreted from the body provides a unique type of non-invasive, anonymized mass test. With the correct workflow, getting from waste to data can be done relatively simply and—importantly—universally for biological contaminants.
- Step 1 is sampling in the treatment plant: An automatic sampler begins by collecting samples, with a mobile variant in use upstream.
- Step 2 is concentration of target organisms: The sample is concentrated according to which organisms/pathogens are being investigated. Subsequent to electronegative filtration, the virus fragments and particles are removed from the filter membrane via homogenizer.
- Step 3 is sample isolation in the lab: Pipetting robots are the best suited instruments for this work. The robots should offer ready-to-use protocols which—in combination with the relevant extraction kits—automatically purify DNA or RNA from the samples.
- Step 4 is detection: Real-time thermal cyclers complete the detection process using fluorescence measurement. Within the polymerase chain reaction (PCR), the targeted DNA or cDNA segment is copied and marked with fluorescence. The more often the series of letters in question appears, the stronger the signal becomes in the real-time PCR process and the clearer it can be determined how high the viral load actually is. Recent studies, however, have suggested that droplet digital PCR (ddPCR) is even better suited to wastewater-based surveillance, as the technique is highly sensitive and more tolerant to inhibitors. Additionally, as innovation continues to expand, technologies like next-generation sequencing (NGS) can also be brought to the forefront of wastewater testing in the quest to identify any new microbe variants that may be circulating.
While SARS-CoV-2 and the COVID-19 pandemic may have shined an initial light on the value of wastewater testing, scientists, researchers, health administrators and others have not shied away from the technique’s potential as other viruses continue to circulate the globe.
For example, on the heels of the ending pandemic, the CDC established four National Wastewater Surveillance System (NWSS) Centers of Excellence to guide public health action during the 2022–23 respiratory disease season—in California, Colorado, Texas and Wisconsin. In all cases, wastewater sampling covered more than 50% of the sites’ population, with Houston, Texas seeing 94% coverage.
Texas
The wastewater sampling area in Houstonincluded 122 sampling sites covering 2.17 million residents. Sampling and testing for SARS-CoV-2, influenza virus and respiratory syncytial virus (RSV) from 48 manholes associated with selected schools provided data to support strategically deployed school vaccination clinics (1,058 COVID-19 and influenza vaccine doses administered), empowered staff members in 48 schools to implement respiratory disease prevention strategies through school reports, and increased public awareness through a dashboard, which recorded approximately 350,000 views as of 2023. Recently, alert notifications were launched (698 registered users associated with 46 schools) to inform users about identification of surges in respiratory viruses.
Colorado
Three Denver sewersheds, covering 1.2 million residents, submitted biweekly wastewater samples for analysis in this project. Retrospective analyses indicated that wastewater sampling detected enterovirus D68 (EV-D68) about 1 month before clinical laboratory signals. This finding led public health officials in Colorado to implement wastewater testing for EV-D68 as part of the enterovirus surveillance model to provide an early warning system for health care surge planning during respiratory virus season.
California
In California, analysis included daily to weekly sampling of 98 sewersheds to detect SARS-CoV-2 variants, RSV, and influenza virus in 41 counties, covering approximately 26 million residents. The results were communicated via dashboards and weekly reports to support local health department decision-making, as well as provide tailored metrics and messaging to communities, providers, and health care systems.
Wisconsin
Wisconsin performed daily to weekly sampling for SARS-CoV-2 at 43 sampling sites covering approximately 2.93 million residents. Data were shared on a public dashboard with alert notifications, a genomic sequencing dashboard and weekly reports to local health departments and water treatment utility companies. Wisconsin’s genomic sequencing dashboard has become an important tool for identifying and monitoring SARS-CoV-2 variants in wastewater, in some cases identifying variants (e.g., BA.5 and XBB) before detection through clinical surveillance. Additionally, 20 sites covering approximately 2.48 million residents were sampled for influenza viruses and RSV. Wastewater concentrations for these viruses were highly correlated with emergency department visits in Wisconsin during 2022–23, forming the basis for continued monitoring through the 2023–24 respiratory disease season.
Detection of opioids and novel psychoactive substances
Prior to the pandemic, wastewater epidemiology was commonly used to detect and track the use of opioids and novel psychoactive substances at a community level. However, the technology didn’t receive much attention, until wastewater sampling more than proved its worth during COVID-19. Once the pandemic was officially declared over, many researchers took wastewater epidemiology back to its roots— drugs of abuse.
For example, just last month, wastewater surveillance in Australia revealed a 75% increase in the detection of the synthetic stimulant pentylone across the country. Pentylone (street name bath salts) is a highly potent and unpredictable synthetic cathinone, producing similar effects to stimulants such as methamphetamine or MDMA.
In the new study, published in Science of the Total Environment, researchers analyzed wastewater from across Australia to determine what type of NPS were being used during a single year timespan with collection dates from February 2022 through February 2023. The study was unique in that the sample intentionally avoided special events and holiday periods to determine “more typical” trends across the year.
Of the 59 different NPS Emma Jaunay and her team looked for, they found 20 in the wastewater samples. While some were found only occasionally across Australia, others were detected at every site for multiple collections—including pentylone. The research team says pentylone displaced eutylone in 2022/2023, highlighting the constantly evolving nature of NPS and how quickly drug preferences change. Still, eutylone and phenibut—two synthetic cathinones similar to pentylone—were also commonly detected in the wastewater samples, according to the study results.
Australia has long been a leader in utilizing wastewater epidemiology for opioid detection. In December 2023, the U.S. launched its first such program, following a design similar to Australia’s previous efforts.
For the program, the National Institute on Drug Abuse (NIDA) partnered with Biobot Analytics to conduct anonymous, population-level wastewater-based monitoring on drugs of use and misuse, and overdose reversal agents. The program includes 70 wastewater treatment facilities and their associated public health departments. Participants span 43 states and 62 counties representing approximately 35 million Americans, and includes a diverse mix of urban and rural communities of varying sizes.
The sites in the program will continue to collect samples once every two weeks through the end of August 2024, with the potential to extend the program if this pilot is successful. After scientists test and analyze the samples, they will share the results with local public health agencies, where the data will add to existing systems for tracking substance abuse.
For example, if the data show increasing amounts of fentanyl in a county’s waste, officials may encourage people who use drugs to employ fentanyl test strips to check for the opioid’s presence before use, or perhaps even increase the availability of Narcan in the region.
Disease spread
In July 2022, the state of New York was still collecting and testing wastewater samples for COVID-19 when they discovered something unexpected—another virus, one that hadn’t been seen in some time. Wastewater in Rockland County—about 30 miles north of NYC—tested positive for polio. Since sampling and testing was ongoing due to the pandemic, the CDC was able to analyze both old and new samples, detecting traces of the polio virus dating back to early June—when the man was first hospitalized with a then-unidentified illness. Positive wastewater samples were then detected in neighboring Orange County, Sullivan County, Nassau County and New York City.
At the beginning of this year, the University of Ottawa launched wastewater monitoring program to detect and prevent the spread of tuberculosis (TB) in Nunavut. The five-year research study aims to develop and optimize TB wastewater monitoring in the City of Iqaluit to determine if earlier detection can allow for earlier public health interventions to treat the disease and reduce transmission. This study builds upon the current TB program in Nunavut, complementing the effectiveness of TB diagnostic methods such as skin tests, sputum tests, and chest X-rays. While these methods continue to successfully identify TB, the new study aims to establish another layer of early detection to these existing strategies.
“We hope to apply the successes of respiratory virus wastewater surveillance around the world to TB, and specifically to TB wastewater surveillance in Nunavut. It is our hope that we will develop the science necessary for Iqaluit’s wastewaters to tell us when TB has the potential to start spreading in the community so that it can be stopped,” said Robert Delatolla, professor in the department of civil engineering at the University of Ottawa.