Testing sewage to halt a pandemic

For the past two years, the COVID-19 virus has been playing a game of cat and mouse with the human race. To beat this virus - and help prevent the next one - a collective effort to ramp up global genetic surveillance is needed to spot the appearance and spread of new variants. One of the most promising and effective ways to monitor disease among a mass population is proving to be digital PCR in wastewater-based epidemiology (WBE).

To appreciate the potential of this method, let's take a journey back to the early months of the pandemic in March 2020. Researchers in China had just announced they could detect the SARS-CoV-2 virus in the feces of infected people. But what happened after the waste was flushed away to a wastewater treatment plant, where it mixed with the outflow from hundreds of thousands of toilets, sinks and drains? Were its genetic remnants still detectable?

With testing methods for COVID-19 still being developed, scientists were eager to find new ways to track cases to understand the scope of the pandemic. Dr. Warish Ahmed, a senior research scientist at the Commonwealth Scientific and Industrial Research Organization (CSIRO), Australia’s national science agency, saw a tweet from a University of Queensland researcher asking if anyone in Brisbane, Australia, could test for SARS-CoV-2 in raw sewage.

Pool-testing 600,000 people at once

Ahmed, whose lab specializes in microbial source tracking in water, otherwise known as MST, had long handled a variety of viral environmental samples from across Queensland. At the first reports of COVID-19 spreading outside of China, he had started work on an analytical method to detect SARS-CoV-2 RNA in wastewater, anticipating the value of an early detection system. The traces were bound to be minuscule when the number of cases was low, but he believed his highly sensitive molecular method could still detect them in raw sewage.

Ahmed tweeted that they should get in touch. Later that afternoon, three researchers from the University of Queensland rang him. As part of routine drug monitoring, hourly samples were already being collected at treatment plants handling wastewater from Brisbane, Queensland’s capital. From these, they created 24-hour composite samples, each murky container representing hundreds of thousands of people. All they needed was the test.

The samples arrived at the CSIRO laboratory the next day. After concentrating and then extracting the tiny amounts of RNA from the raw sewage, RT-qPCR tests found what they were looking for: faint but definite traces of SARS-CoV-2 RNA. Using a simple model, they roughly estimated that 171 to 1,090 people in the catchment area, which covers about 600,000 people, were potentially infected. It was the first confirmed evidence of SARS-CoV-2 RNA in wastewater in Australia, and, says Ahmed, “the results were proof this method could potentially be used to identify hotspots and infected people.” They had struck upon what might prove to be a cost-effective way to pool-test entire communities for the disease. When Ahmed and his colleagues published their proof-of-concept research, it created ripples in the scientific community.

The future of WBE and COVID-19

Despite the availability of vaccines, there is still a need for public health agencies to survey COVID-19 circulation on a mass – and affordable – scale. “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,” Ahmed says. “If you monitor wastewater, you can potentially screen 100,000 people by analyzing one sample.” Metcalfe adds that "our objective is to basically pick up one infected from one hundred thousand people."

When the COVID-19 infection numbers are finally reduced, it will become harder to detect in the wastewater, as the viral concentration will be very low. Their solution, Ahmed says, “is incorporating other sensitive detection methods – for example, the QIAcuity.” QIAGEN’s QIAcuity nanoplate digital PCR system is designed to find such faint genetic signals. It partitions the sample into 26,000 individual reactions, making it far easier to pinpoint a single positive. Ahmed says, “We believe digital PCR is going to increase the sensitivity at least five- to ten-fold.

Australia is not the only country using this method. Surveillance testing allows public authorities to collect data from broad sweeps of the population, including people who are not reflected in public-health statistics because they lack access to healthcare. Currently, 70% of all US states are using QIAcuity for ultra-sensitive wastewater detection of SARS-CoV-2 infections, for example. And as COVID-19 is becoming endemic, testing needs will increasingly shift from detecting viruses to monitoring the spread of infectious diseases.