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Wastewater tells a story

Beneath its murky and foul-smelling appearance, wastewater has a value that goes far beyond its recycling and possible reuse. It is an environmental and health indicator that contains different pollutants from industrial and especially urban sources, something that makes wastewater a source of information on human exposure to various chemical compounds and pathogens.

The tool that translates its language is wastewater based epidemiology (WBE), originally linked to the study of the presence of the poliovirus, whose monitoring is today a useful instrument to alert about the circulation of wild strains or neurovirulent vaccine strains among the population. For the rest of the viruses, its application has been relegated to research projects; however, the current COVID-19 pandemic has boosted the study of SARS-CoV-2 in wastewater worldwide, making it a major early warning tool, with a promising future.

“It travels along the great tracks of human intercourse, never going faster than people travel, and generally much more slowly”. This is how John Snow (1813-1858), English physician – not Jon Snow, a character from Game of Thrones – described the behaviour of cholera bacteria when they swept through the city of London during the most violent outbreak in England. Contrary to the “miasma” theory – toxic vapours emitted by decomposing soil matter and impure water – which prompted the authorities to empty all the city's cesspits and septic tanks into the River Thames, Snow proposed that the spread of cholera was due to water contaminated by a "morbid matter" invisible to the human eye

Published in SWM Bimonthly 11 - February 2022
SWM Bimonthly 11

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.

Wastewater based epidemiology serves as a tool for the detection of SARS-CoV-2 in the population

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.

  • Untreated sewage is a global problem that puts the health and well-being of millions of people at risk as a route for the spread of disease
    Untreated sewage is a global problem that puts the health and well-being of millions of people at risk as a route for the spread of disease

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.

Tracking down SARS-Cov-2

Raw sewage is an ideal ecosystem to assess viral diversity; as well, it provides a rich environment for the growth of diverse host species and thus their viruses. According to a pioneering study in the application of metagenomics - the study of the genome of microbial communities - to sewage viruses published by the journal mBio in 2011, carried out by the UB Food and Water Contaminating Viruses Laboratory and the University of Washington, raw sewage provides a rich matrix not only to identify novel viruses, but also to study the diversity of known viruses.

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.”

"WBE stands out as the most important tool missing in the armamentarium to control COVID-19", Dr. Christian Daughton in Smart Water Magazine

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."

  • WBE has become a major early warning tool to monitor the presence of SARS-CoV-2 in the population
    WBE has become a major early warning tool to monitor the presence of SARS-CoV-2 in the population

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.

The results can contribute to the establishment of an efficient epidemiological surveillance system for SARS-CoV-2 in the future, in order to learn about the evolution of the global infection rate in a community, and to detect early any potential outbreaks. "If we have the technology to detect SARS-CoV-2 genetic material, which we do, and we are able to correlate the data in wastewater with clinical epidemiological information, we can have a very useful tool that also provides real-time information about a large anonymous population to monitor the pandemic in the communities under study", he explains. Beyond COVID-19, the project will lay the foundation, as well as advantages and limitations for future applications of wastewater analysis in disease control.

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.

"We are able to sequence, that is, to know the genetic code of all the virus variants that appear". COVIDBENS has also served as an early warning of possible outbreaks and has shown that it is capable of anticipating detection up to eighteen days ahead of the data reported by the Spanish health system: "In addition to the specific detection of the virus, we use standards to extract data on what the viral concentration is. It is not enough for us to know if the virus is present or not, we also quantify it".

Detection of SARS-CoV-2 in wastewater

The analysis of wastewater for epidemiological purposes is based on the excretion products of human metabolism that arrive intact at WWTPs. In the case of SARS-CoV-2 detection, although there are different protocols designed for the detection of the virus in wastewater, they have several steps in common: (1) concentration and precipitation of the sample; (2) extraction of the genetic material (RNA); and (3) detection of the SARS-CoV-2 specific genetic material.

In turn, for each of these steps, there are different analytical methods, commercial kits or detection techniques, all of them with their pros and cons. In the particular case of detecting the genetic material of the virus in wastewater, the most widely used technique is the real-time quantitative polymerase chain reaction, usually known by its acronym RT-qPCR.

It is a technique that stands out for its versatility, sensitivity and efficiency, and is suitable for the analysis of wastewater.

Much more than waste

The COVID-19 pandemic has not only highlighted the vital importance of sanitation, hygiene and adequate access to clean water to prevent and contain the disease, as well as a water sector essential for all of the above to occur, but also it has drawn attention to wastewater as much more than a waste to be treated. Wastewater has been a near-perfect mirror of SARS-CoV-2 prevalence in the population and its evolution, underscoring the enormous potential of WBE as a key tool to help contain and mitigate COVID-19 outbreaks while minimizing global ripple effects on public health and safety, accessible healthcare, food security, the stability of economies and financial institutions. "Investing in a permanent wastewater based national epidemiological network could prove vital in preparing for more rapid and effective control of the next inevitable pandemic", says Dr Daughton.

Wastewater has been a near-perfect mirror of SARS-CoV-2 prevalence in the population and its evolution, showing the potential of WBE

That is why this pandemic, notwithstanding its negative health, economic and environmental consequences for societies worldwide, provides a significant opportunity to prove that, as an emerging discipline, WBE has great potential - combined with commonly applied epidemiological studies – for the detection and management of disease transmission in the population. The presence of a substantial amount of viral RNA in faeces and urine implies the possibility of tracking disease occurrence and transmission trends over time using this methodology. "WBE applied consistently and thoroughly could become a very suitable early warning system for potential disease outbreaks. But it should not be forgotten that it is a complementary tool. It cannot replace classical epidemiological studies, based on monitoring clinical cases, but rather complement them. The combination of both approaches is what will allow progress to be made in the monitoring of this and other future epidemics/pandemics," explains Félix Hernández.

  • As an emerging discipline, WBE has great potential to be used in the detection and management of infectious disease transmission
    As an emerging discipline, WBE has great potential to be used in the detection and management of infectious disease transmission

Currently, environmental surveillance for the presence of SARS-CoV-2 in wastewater is already used in approximately fifty countries and more than a thousand cities. COVID-19 WBE Collaborative gathers this information on its website, which aims to empower collaboration on a global scale for wastewater based epidemiology of SARS-CoV-2. "The main challenge for sewage surveillance is that the water sector is not accustomed to providing data for the health sector and the health sector is not used to consider environmental surveillance when looking at public health," says Dr Medema. A relationship - that of the water sector and the health sector - that has been greatly strengthened by the pandemic and that in the future could effectively serve as an early warning tool for future outbreaks or pandemics, for health management and as a safety net against possible undetected sources of infection.

Gloria Sánchez (IATA-CSIC): “WBE is here to stay. We have to take advantage of the infrastructure and networks that have been created"

"The water sector and the health sector, and all surrounding organisations, need to communicate efficiently and in real time the information so that the measures taken are effective. Wastewater based epidemiology applied to COVID-19 is a tool that is here to stay. We have to take advantage of all the infrastructure and networks that have been created," emphasizes Gloria Sanchez. However, as Margarita Poza points out, "without funding you can't do research. To implement it permanently, we need funding for infrastructure and for research staff”.

The enormous potential of wastewater based epidemiology has been shown in multiple scientific studies carried out internationally and, although more research is needed – and more funding for it – wastewater will continue to tell us even what we cannot see. Let's listen to it.