Wastewater analysis for population health surveillance

Awareness of the utility of wastewater for epidemiology was raised during the recent COVID-19 pandemic. CLI caught up with Dr Andrew Singer (UK Centre for Ecology & Hydrology) to find out more about the development of wastewater-based epidemiology and what it can do for us.

Many people will have only become aware that wastewater is monitored for the presence of infectious disease agents during the COVID-19 pandemic or with the news that poliovirus has been found in London’s wastewater. Can you give a brief description of wastewater monitoring and how it has taken off recently?

Wastewater-based epidemiology (WBE) is experiencing a renaissance; it is the use of wastewater for the determination of health attributes of the sampled population, often in a semi-quantitative manner. Similar concepts have been employed for over 150 years in the detection of typhoid and cholera, with Dr John Snow’s track-and-trace efforts confirming contaminated water from the Broad Street water pump as the source of the 1854 cholera outbreak in London, England being among the more well-documented examples. For nearly 40 years, wastewater samples have been collected across the globe to monitor the progress of the World Health Organization’s Global Polio Eradication Initiative. This is because those infected with poliovirus will shed the virus in their stool, which invariably enters the wastewater system. Hence, recovery of poliovirus from wastewater is a ‘red flag’ that there is a risk of poliovirus transmission. Homology of the recovered poliovirus RNA to the vaccine-derived poliovirus type 2 in London, for example, would suggest that vaccination is the source of poliovirus, which was imported from abroad (where that vaccine is still used), and not evidence of human-to-human transmission. However, the transmission risk is assessed based on the number of mutations or degree to which recombinations had occurred in the sewage-derived poliovirus. The more divergent the recovered poliovirus is from the vaccine strain, the more concern it raises for public health officials. This played out most recently in London in early 2022 under the polio surveillance programme conducted by the National Institute of Biological Standards and Control; similar poliovirus RNA was observed in wastewater from Israel and the United States later this year.

Concurrent with COVID-19 and poliovirus detection in wastewater, a new threat was identified and similarly monitored in wastewater, monkeypox. Several programmes are ongoing for monkeypox surveillance, generating highly informative data on the spread of the virus and its penetration into nearly all watersheds of the world, in which such surveillance is active.

How did wastewater surveillance transition from a tool to observe the eradication of poliovirus to a global pathogen surveillance tool?

WBE had been a topic of research by a small subset of the academic community for decades – maintained by the need to monitor poliovirus, but with aspirations to explore what else could be done with it to inform public health officials and governments on population health and disease prevalence. Researchers were focused on developing methods for environmental monitoring for antimicrobial resistance (AMR), often from wastewater, such as skills and methods for wastewater sampling, RNA and DNA extraction, quantification (usually by qPCR), and most recently, sequencing. Although all of these tools and methods were widely known in 2009, WBE was not employed to track the progress of the swine flu pandemic. The most likely reason for it not being used during the influenza pandemic was because doctrine stated that influenza is a respiratory virus and, as such, should not be present in the stool.

And then the COVID-19 pandemic happened.

The COVID-19 pandemic created a WBE renaissance. In early 2020, many in the field of environmental microbial surveillance with ready access to wastewater examined the feasibility of recovering from sewage the RNA of the virus that causes COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To most of us, it was surprising how easy it was to find the coronavirus in wastewater considering the fact that SARS-CoV-2 is a respiratory pathogen and was not expected to be in the stool or urine, let alone in significant enough quantities to allow for detection once diluted in thousands of toilet flushes. The doctrine states that DNA can survive reasonably well in the environment, but RNA is highly labile. Yet, it was clear, SARS-CoV-2 RNA was readily recoverable from wastewater – the most hostile environment imaginable for a pathogen that is not designed to transmit through a fecal oral route and certainly an incomprehensibly hostile environment for labile RNA. The apparent paradox of recovering SARS-CoV-2 RNA from wastewater, it is argued, might be possible through the protective effects of the matrix in which the virus is nested – poo! The fact that nearly all of the virus is excreted within the stool, it follows that as the stool is broken down into smaller and smaller fragments, the RNA is increasingly more likely to be exposed, yet, much of it (or should I say ‘enough’) appears to remain protected in what might be sub-micron-sized poo particles. These particles, in theory, become the RNA signal used by those conducting WBE.

The feasibility of employing WBE for the determination of the prevalence of SARS-CoV-2 in a region was documented by a Dutch team of scientists by the end of March 2020, leading to the rapid expansion of such programmes at local and national scales across the globe. Early successes were found on university campuses, where validation of the method was often more achievable because of obligatory mass testing regimes. Hence, when a surveillance team recovered SARS-CoV-2 RNA from the wastewater outside of a hall of residence, the campus authorities were able to mass-test all the residents. As COVID-19 can manifest asymptomatically – and so the carrier does not necessarily know they are infected – passive surveillance systems, such as WBE, played a crucial role in establishing a non-invasive, low-cost tool for population surveillance, uniquely capable of detecting symptomatic, asymptomatic and pre-symptomatic COVID-19 cases.

How is WBE data used to inform on population health?
During the pandemic we explored many different use cases for WBE. Sampling at a sewage treatment plant would be indicative of the widest possible population in a region or city. Other use cases included primary and secondary schools, universities, prisons, care homes, airports, aeroplanes, ferries, truck stops, city manholes, and large businesses.

The genome copies (gc) of SARS-CoV-2 RNA was the unit of measure, which was invariably normalized to the volume of liquid from which it was extracted, i.e. 1000gc/L. But many WBE practitioners also explored ways to normalize genome copies to the flow of sewage from which it was recovered, which could be represented as a load of virus per day. Determination of viral loads were often infeasible due to the lack of suitable tools for measuring flow in the majority of locations from which samples were acquired. Alternative methods for determining load were to normalize to other chemical or biological markers that are reliably present in (nearly) all toilet flushes. There is an ongoing debate about which normalization procedures are best for estimating prevalence of SARS-CoV-2 in wastewater. Despite some markers being highly useful for some groups in some sewage catchments, many other teams had little success with normalization and proceeded to simply collect reasonably comparable samples from the same location to generate a longitudinal dataset that focused on the slope of the 3-day rolling average – a positive slope means virus numbers are increasing, with a negative slope indicating declining numbers.

The benefit of a longitudinal dataset is that the lack of precision associated with any one sample is compensated for by redundant samples, i.e. if you sample 7 times per week and one of the samples yields erroneous results, the trend should still be apparent. Hence, most use cases for WBE focused on the direction of any trends over time, i.e. the number of COVID-19 cases in a region appears to be going up and the rate of increase is steeper than was witnessed back in time period X, Y or Z.

What other applications for WBE are emerging?
As the WBE methodology for SARS-CoV-2 became more routine and confidence grew in the data and the trends it revealed, many teams began to explore how the extracted RNA could be used to reveal trends about other RNA viral targets, such as: respiratory syncytial virus (RSV), influenza A and B, human norovirus, and hepatitis A virus (HAV). The value in determining the presence and prevalence of additional pathogens in the same sample used for SARS-CoV-2 detection was to generate greater value from the same effort. Considerable effort is now being placed in developing methods that can leverage the pipelines developed for sample preparation during the pandemic for not only RNA targets, but also DNA targets: bacteria and fungal pathogens, as well as specific genes, such as antimicrobial resistance.

What is one of the greatest barriers to wider uptake of WBE?
The majority of the cost of conducting WBE is associated with a person or people visiting a site to collect a wastewater sample and couriering it back to a laboratory for analysis. Due to this cost, it is impractical to scale WBE for national pathogen surveillance for any great length of time. Considerable interest is being focused on how these costs can be reduced to enable wastewater surveillance at scale. A national surveillance network that relied on an automated end-to-end solution for generating a rich dataset of pathogens and antimicrobial resistance genes would offer significant value to public health monitoring, at a fraction of the cost of traditional mass testing, and in near real time. In fact, there is no comparable dataset to one that could be collected through wastewater. Such data would empower public health decisions to be made rapidly, and result in government actions leading to the termination of epidemics and pandemics before they ever really get started. There is equal potential to include chemical sensors in such automated end-to-end wastewater sampling solutions, further expanding the range of questions that could be asked about the health and behaviours of the population. Increasing target breadth brings greater value from the same investment – making it easier for those with limited capital to justify a WBE programme.

The future of WBE is exciting and could include automated sequencing pipelines identifying previously uncharacterized microbial threats. The implementation of a such a surveillance tool at airports and other ports of entry across the globe can be the basis of a truly integrated and networked global pathogen surveillance network of the future.