Sewage as a health indicator

Detecting diseases in wastewater

Analysing DNA or RNA in sewage can provide information about public health. Researchers at Delft University of Technology are working on a new technique based on measuring all the protein in sewage samples, whether from humans or microbes. Like DNA and RNA, proteins can also serve as an indicator for detecting emerging diseases.

TEXT: MARLIES TER VOORDE

Through urine and faeces, people excrete proteins, viral remnants and microbes, all of which end up in the sewers. Just as a tube of urine contains information about an individual’s health, a sample of sewage contains information about the health of the group of people using that sewer system. In the Netherlands, this involves over three hundred different groups – that is the number of sewage treatment works (STWs) spread across the country.

Sewage contains indicators of various diseases. The National Institute for Public Health and the Environment (RIVM) has therefore been conducting sewage monitoring for over thirty years. For example, in 1992 the research institute found traces of the poliovirus in certain regions, and since 2020 the laboratory of the RIVM’s National Sewage Surveillance Programme (NRS) has been analysing wastewater for the concentration of coronavirus particles (see box: Coronavirus wave forecasts).

Researchers at Delft University of Technology are now expanding wastewater monitoring with a new method. Rather than focusing on small molecules, DNA or RNA, as conventional methods do, this method focuses on proteins in the wastewater that may originate from humans or microbes. De novo metaproteomics: this is the name of the method, on which the researchers published a scientific article last January in the journal ISME Communications (see box ‘What is metaproteomics?’).

A welcome addition

De novo metaproteomics could be a valuable addition to the sewage surveillance programmes already in place, says Martin Pabst, a researcher in environmental biotechnology and head of the metaproteomics research group at Delft University of Technology.

‘Thanks to such surveillance programmes, doctors can see whether certain campaigns or treatments are proving effective, and whether an infectious disease is flaring up or, on the contrary, dying down.’ This can also be determined through other studies, but these are often difficult to carry out and expensive because they require a large number of people to be approached and tested. This is often done using blood tests or questionnaires.

With de novo metaproteomics, any disease that produces specific proteins or unique microbes (biomarkers) can be detected. ‘And the method is very cheap,’ says Pabst. ‘Sewage water is collected anyway, and the analysers are already in place. The results are available within a day. We can then track these results on a dashboard, for example, similar to what was developed for COVID-19 at the time. Policy-makers can then clearly see regional trends and are alerted if something is amiss.’

Possible incidents that could be detected in this way include, in addition to outbreaks of infectious diseases, unnoticed exposure of people in a particular area to chemicals or industrial pollutants that cause illness. ‘That might not happen so quickly in the Netherlands,’ says Pabst, ‘but there are also countries where environmental regulations and safety standards are much less strictly enforced.’

Wide diversity of proteins

During the coronavirus crisis, the viral concentration was determined from RNA present in the sewage. Pabst believes that, at the time, it would probably not have been possible to foresee the waves of infection using de novo metaproteomics. The new method is therefore still under development. ‘It’s all about sensitivity,’ says Pabst, ‘and that increases with the amount of data.’ Because the diversity of proteins is much greater than that of genes, it is actually more difficult to identify a species using proteins than using DNA or RNA.

With the RNA method, the signal being sought is significantly amplified. ‘This makes it a very sensitive technique, which is a major advantage. But you do need to know in advance what you’re looking for,’ says Pabst. ‘Consequently, you may overlook new variants of a virus. And because of the technique’s sensitivity, false positives can also occur: you measure something, but it turns out there is actually nothing wrong.’

The new method his group is working on is not targeted: it measures all the proteins present, whether they are being sought or not. And because no signals are amplified, there is less chance of false positives.

Value for healthcare

De novo metaproteomics is a technique still under development and is therefore not yet being used for large-scale analyses. However, in a proof-of-concept study, Pabst and his team analysed wastewater samples from Utrecht and from the Harnaschpolder wastewater treatment plant in Den Hoorn, near Delft. During this campaign, which took place over three winter months, they detected around two hundred human proteins in the wastewater. These included substances associated with cancer, as well as indicators of virus-induced inflammation and antibodies.

‘It’s great to see that so much is being discovered in sewage,’ says Gertjan Medema, a researcher at KWR and one of the driving forces behind the coronavirus surveillance in the sewer system six years ago. He is not involved in the research in Delft. Medema: ‘The fact that wastewater also contains proteins linked to our health is a topic that various institutes are currently working on. Delft University of Technology is one of them.’ Medema sees this primarily as exploratory research, focused on what can be found in sewage. According to him, applying this information in healthcare will require a number of further steps. ‘To do this, we need a better understanding of the relationship between the presence or concentration of certain proteins in sewage and the prevalence of a disease in the population.’

More research is also needed into the stability and behaviour of proteins in sewage, says Medema. This is because some human proteins that are common in faeces or urine have not been detected in sewage at all.

Medema: ‘Researchers have so far focused on what can be observed in sewage. The next step should be to determine how this information can be used to improve healthcare.’ Pabst agrees. ‘At the moment, what is needed above all are more large-scale studies, in which we monitor large cities over a few seasons. Then we can find out whether the data from the sewage correlates with medical data on the population, validate the method, and identify which biomarkers are most useful for us to focus on.’

Given that mass spectrometers are becoming more sensitive every year, Pabst believes that de novo metaproteomics could, in about five years’ time, be a fully viable supplementary method for analysing sewage water – for example, to monitor the population’s immunity and vaccination rates in real time.