Wastewater tells a story

Snow proposed that the spread of cholera was due to water contaminated by a "morbid matter" invisible to the human eye

This "morbid matter" responsible for the acute diarrhoea that characterizes the disease – abundant, watery, rice-washing water-like stools – was transmitted among the London population through the sewage that turned the Thames into a "deadly sewer", as Charles Dickens defined it in his novel Little Dorrit (1857) years later. The fact is that London at that time was very far from the sanitation standards and notions of environmental hygiene that we enjoy today, and the same waters of the Thames that had been contaminated with the contents of the cesspits and septic tanks of the city were used again to supply drinking water to the city through two water companies. Although the study of diseases as population phenomena dates back to the pestilential fevers that devastated the population on the banks of the Nile River around 2,000 BC, John Snow is today considered the father of modern epidemiology for laying the theoretical and methodological foundations of epidemiology by relating, in London in 1854, mortality rates to population behaviour with respect to the water supply.

Although this problem in London dating two centuries ago seems very distant, cholera is just one more example of the disease that can be transmitted through water. According to the Safe water, better health report published by the World Health Organization (WHO) in 2019, after diarrhoeal diseases, which are the most common, there are at least twelve other diseases in the world directly linked to water due to its use for drinking, sanitation and poor hygiene (WASH). Hepatitis A, typhoid fever, polio, malaria, schistosomiasis and dysentery are some of the diseases that kill millions of people worldwide. The UN also warns that more than 80% of the wastewater resulting from human activity is discharged into rivers or the sea without any kind of water treatment, causing water pollution.

This relationship between water and people's health highlights how essential it is not only to have water treatment systems that apply the necessary physical, chemical and biological processes to the resource for its reuse or for its return to natural water bodies, but also to have monitoring protocols that provide sufficient data to estimate the dimension and possible causes of spatio-temporal variations in water quality that affect public health. As former WHO Director-General Dr Lee Jong-wook (1945-2006) wrote: "Water and sanitation are one of the main drivers of public health. I often refer to it as 'Health 101', which means that once we can secure access to clean water and adequate sanitation facilities for all people, irrespective of the difference in their living conditions, a huge battle against all kinds of diseases will be won."

Untreated sewage is a global problem that puts the health and well-being of millions of people at risk because it is a pathway for disease transmission, including some viral diseases. This is not the case with SARS-CoV-2, the virus that causes COVID-19. Currently available information, according to the World Organization for Animal Health (OIE), suggests that it comes from an animal, and that there is still not enough scientific evidence to explain the original route of transmission to humans. On this point, a study by the Dutch National Institute of Public Health and Environment (RIVM) published in the journal The Lancet Gastroenterology and Hepatology points out that it is still unclear whether SARS-CoV-2 is viable in environmental conditions that could facilitate faecal-oral transmission. Moreover, researchers from the University of Granada in Spain carried out an epidemiological analysis of 134 workers at 59 wastewater treatment plants (WWTP) in the Andalusian province of Granada – critical urban water cycle infrastructure and, therefore, essential in the elimination of SARS-CoV-2. The results, published in the International Journal of Water Resources Development, revealed the seroprevalence in this group was similar to the incidence rate found in the general population in the province. This suggests there is no significant transmission of the coronavirus through wastewater because, like other coronaviruses, SARS-CoV-2 is not as resistant to environmental conditions in wastewater as, for example, noroviruses.

During the pandemic, it was discovered that SARS-CoV-2 was also excreted in faeces, ending up in sewage, and that viral RNA was detectable in municipal wastewater. This finding raised questions about the potential health hazards to people who were directly or indirectly exposed to sewage - as mentioned above, there is no significant transmission - but it also provided opportunities to use sewage to monitor trends in virus circulation in the community, allowing entire populations to be assessed at a glance.

The low probability of detecting asymptomatic people put the focus on WBE to determine the burden of undiagnosed infections

Environmental virology linked to wastewater dates back to the 1940s and the efforts to detect the polio virus. Since then, cell culture methods useful to detect enteroviruses have been replaced by molecular biology and analytical chemistry techniques to detect pathogens. Today, thanks to the application of analytical sciences, it is possible to know the human exposure to various chemical compounds or pathogens - such as viruses - through wastewater. This is known as wastewater based epidemiology (WBE). Its initial application, and the one that is best known and established today, was to estimate illicit drug consumption in populations, as well as legal substance abuse (alcohol, drugs or tobacco), with the COVID-19 pandemic it has become the main early warning tool to monitor the presence of SARS-CoV-2 in the population. Dr Christian Daughton, a researcher now retired from the U.S. Environmental Protection Agency and a pioneer of WBE more than two decades ago, says that “The utility of WBE for monitoring the incidence and spread of community-wide infectious disease (such as Covid-19) is currently much more limited than the application of WBE for determining community-wide usage of anthropogenic chemicals (such as drugs) or exposure to naturally occurring xenobiotics.”

Based on Daughton's idea of imagining wastewater treatment plants as a tool not only to track an emerging class of contaminants, but also to monitor the general state of health in the entire community, numerous research groups around the world are studying the presence, characteristics and epidemiology of different viruses based on their detection in wastewater. The development of molecular technologies applied to environmental studies has shown that even in highly industrialized countries there is a high prevalence of viruses in the environment, which causes a significant impact on public health and important economic losses, mainly through the transmission of viruses through water and food. Gertjan Medema, Principal Microbiologist at KWR Water Research Institute in the Netherlands, has been analysing the transmission of infectious diseases and antimicrobial resistance through water systems for ten years and how this can be prevented by technical and non-technical management measures. With the outbreak of the pandemic, his research team was the first to think that they could find SARS-CoV-2 in wastewater, coming from the faeces of infected people: “Testing wastewater can complement information from conventional testing – PCR, antigen and serological tests – by allowing authorities to monitor the occurrence of the virus in large populations over time”.

Source: Randazzo et al., 2020

In the midst of pandemic uncertainty, in March 2020 no one had sufficient capacity to know the true number of infected people. The low probability of detecting asymptomatic people and those with mild symptoms during clinical surveillance prompted researchers to focus on WBE to determine the burden of undiagnosed infections at the community level, which has proved critical in refining estimates of case fatality rates. Researchers from Cranfield University (UK) and the Chinese Academy of Sciences developed a paper-based device to detect the coronavirus in wastewater. It is a small analytical tool with different functional areas printed with a wax printer that integrates all the necessary processes for nucleic acid testing in inexpensive paper material. "Currently, the test is designed as a rapid and cost-effective diagnostic tool for the mass population rather than for individuals," explains Dr. Zhugen Yang, Lecturer in Sensor Technology at Cranfield Water Science Institute, whose goal is to implement it permanently in epidemiological surveillance: "We will develop inexpensive sensors to rapidly map hotspots, which will enable analysis requirements to be met."

In Spain, the Agrochemistry and Food Technology Institute (IATA-CSIC) and the Segura Soil Science and Applied Biology Centre (CEBAS-CSIC), who collaborate with Esamur (the Sanitation and Wastewater Treatment Authority in the region of Murcia) since 2016 on a quantitative risk analysis focused on the effectiveness of WWTP treatments in order to identify pathogenic bacteria, enteric viruses, Clostridium spores or disinfection by-products, were the first to validate a methodology to detect SARS-CoV-2 RNA in raw wastewater. "The concern arose at the end of February following publications at the international level revealing the presence of RNA traces from SARS-CoV-2 in faeces, which led to the thought that viral particles could accumulate in wastewater from cities", reveals Ana Allende, researcher at CEBAS-CSIC.

The implementation of this validated and rapid methodology was possible thanks to IATA's experience working on epidemiological surveillance of viruses in water

Thanks to IATA's experience working on epidemiological surveillance of viruses in water, it was possible to implement this validated and rapid methodology that allowed detecting the concentration of SARS-CoV-2 in wastewater before the first clinical cases were recorded in three of the six Murcian municipalities where sampling was carried out. "We have been working for many years and developing molecular techniques for the detection of viruses that, traditionally, are transmitted through faeces and can therefore end up in food, such as gastroenteritis viruses or noroviruses, but also more important viruses such as hepatitis A or hepatitis E", comments Gloria Sanchez, senior scientist. She explains the validation procedure they followed: "With a strain of porcine coronavirus, which has physicochemical characteristics similar to SARS-CoV-2 – mainly the lipid envelope –, and with a mengovirus, we doped water samples that we had stored from previous sampling. The results showed that the procedure we normally used for the detection of norovirus and hepatitis A in wastewater was a procedure that could be used routinely for the detection of SARS-CoV-2 RNA in water." Subsequently, the IATA-CSIC group led a second study in Valencia, in collaboration with I2SysBio, where traces of SARS-CoV-2 were also detected in retrospective water samples from late February 2020.

IATA-CSIC team led by Gloria Sánchez, on the left of the image.

Also in Spain, among the many capabilities of the Spanish Network of Wastewater Analysis for Epidemiological Purposes (ESAR-Net), created in 2017 and formed by different Spanish research groups, work was already underway on new applications such as the study of population exposure to chemical contaminants or the identification of health indicators for a given population. "The rationale is the same as in the case of exposure to pollutants or illicit drug use, among other examples," explains Félix Hernández, professor and director of the University Institute of Pesticides and Water (IUPA). "Everything we are exposed to, including a viral infection, is reflected in wastewater, as long as a series of criteria are met, such as the existence of specific biomarkers that are sufficiently stable in wastewater, and suitable analytical techniques to detect and quantify them." Hernández is currently leading a research project that aims to apply the principles and strategies of wastewater based epidemiology (WBE) to the identification and quantification of SARS-CoV-2 genetic material in wastewater from communities in the province of Castellón.

Based on the effectiveness demonstrated in the detection of SARS-CoV-2 genetic material in wastewater and its possible use as a complementary system to the health surveillance of the pandemic, the Spanish Ministry for Ecological Transition and Demographic Challenge (MITECO) publishes weekly results of the sampling carried out in the more than thirty WWTPs on its website since November 2020. They are part of a project on surveillance and early warning of COVID-19 in wastewater (VATar-COVID-19), defined as a specific application of wastewater based epidemiology to monitor certain substances associated with population habits and lifestyles, and its application in public and environmental health. Also noteworthy in Spain are the environmental monitoring protocols established in different regions such as Catalonia, through Sarsaigua – led by the ICRA – or the Sistema Vigía in Madrid, promoted by Canal de Isabel II.

In March 2021 the European Commission adopted a common approach to establish a systematic surveillance of SARS-CoV-2 and its variants in wastewater in the EU

Also, in light of the progression of the pandemic and the appearance of a second wave in several countries, in March 2021 the European Commission adopted a common approach – implemented in October – to establish a systematic surveillance of SARS-CoV-2 and its variants in wastewater in the EU. The strategy strongly recommends "tracking the presence of the virus in wastewater as a relatively cheap and reliable way to collect essential information", given that the information collected to date has shown a direct correlation between the amounts of virus found in wastewater and the number of infected people in a community.

In this regard, the COVIDBENS project, funded by Edar Bens and carried out in the region of Galicia (Spain) since April 2020, applies a statistical regression model capable of estimating the number of people carrying the virus in the population based on the analysis of the viral load in wastewater. In addition to this, since the beginning of 2021, they monitor and watch for the appearance of new mutations and variants of SARS-CoV-2 in wastewater using massive sequencing technologies. "With this methodology we are able to tell which variants of the virus are in the metropolitan area of the city of A Coruña", explains Margarita Poza, microbiologist at the Institute for Biomedical Research (INIBIC), Associate Professor at the University of A Coruña and leader of this pioneering project.