Something in the Water

Scientists agree that infectious diseases are one of the most critical threats to global public health today (1) – an opinion borne out by COVID-19, monkeypox, and more. To help mitigate the spread of disease, communities have begun to rely more heavily on early detection through wastewater-based epidemiological testing (WBE) using molecular techniques.

Water testing has historically focused on testing sources for the presence of parasites, bacteria, and viruses upstream of human contact – but the COVID-19 pandemic spotlighted the potential of using wastewater as a sentinel system to monitor and predict outbreaks by detecting and quantifying pathogens shed from infected individuals. This type of surveillance enables scientists to detect and quantify small changes in pathogen concentrations in wastewater from a pooled community such as a building, campus, or geographic region. With this information, researchers can potentially forecast ebbs and flows in disease levels and help local authorities prepare accordingly.

The go-to technology for detecting and quantifying nucleic acids is quantitative PCR (qPCR). However, when analyzing low target molecule concentrations, many scientists have found that qPCR is not sensitive enough for wastewater testing and that other techniques are better suited to the task. For example, droplet digital PCR (ddPCR) is a molecular counting technology, related to classic qPCR, that partitions a PCR reaction into tens of thousands of sub-reactions and analyzes each partition for the target molecule of interest. This technique is particularly attractive for wastewater testing because of its precision at low levels, multiplexing capability, and tolerance to inhibition.

Because it’s a direct counting method combined with distribution analysis, ddPCR is not dependent on standards for its precision and accuracy. At low concentrations (a few molecules per reaction), quantification is practically only limited by stochastic effects from sampling.

The partitioning process also simplifies the chemistry surrounding multiplexing. Higher-abundance targets no longer “starve out” lower-abundance targets, so four, eight, or even 12 targets can be amplified and analyzed in the same reaction. This allows for larger panels and the addition of recovery reference targets.

As a post-amplification analysis reaction, ddPCR is not dependent on amplification efficiency for its accuracy and can therefore tolerate moderate inhibition. This is advantageous when testing wastewater samples because, even after purification, they can contain significant inhibitors of the PCR process.

Now more than ever, government authorities are relying on wastewater testing, leveraging molecular techniques to monitor infectious disease outbreaks in our communities. Technologies such as ddPCR permit more refined monitoring and allow efficient, definitive localized surveillance, enabling authorities to predict future surges and enact policies to mitigate outbreaks and save lives.