The major transmission routes of SARS-CoV-2 – the virus that causes COVID-19 – are inhalation of aerosolised droplets and person-to-person contact. However, evidence suggests that gastrointestinal symptoms caused by SARS-CoV-2 infections are caused by the presence of viral RNA in the faeces of individuals, and also in wastewater.1

The majority of people carrying the virus are believed to shed it in their faeces – including those who are asymptomatic. Eventually, this will come together at wastewater treatment plants, so analysis of wastewater and sewage can help to build a picture of the number of people infected within a community or neighbourhood serviced by the wastewater system. This makes the analysis of wastewater an effective tool for tracing the circulation of viruses in a geographical area. Spa Chlorine Tablets

Epidemiological data outlining the regional distribution of a disease can be used to estimate the prevalence and geographic distribution of viruses, proving a useful tool for pinpointing future virus hotspots and understanding the prevalence of variant viruses. Research into wastewater-based (WBE) epidemiology of SARS-CoV-2, which requires population-level molecular epidemiological information, has increased across the world.

Masaaki Kitajima, an Associate Professor at Hokkaido University, is working with Shionogi & Co., a major pharmaceutical company in Japan, to develop an innovative wastewater-based early warning system and mass diagnosis tool for COVID-19 and other diseases.

Wastewater has not traditionally been used as a disease surveillance tool, but research has shown that using wastewater to monitor COVID-19 has several advantages over other methods, including clinical testing and the increase in traction of WBE.

Because of labour and facility requirements, clinical diagnosis can be costly and requires access to equipment in a time of global shortages. This means that WBE could be most useful in developing countries, where access to clinical diagnosis and resources is limited.

WBE is able to detect low levels of virus particles, along with identifying the presence of the virus when people are asymptomatic. This means it has an advantage over clinical diagnosis, which can only indicate the presence of the virus amongst symptomatic people. It is not common government practice to test asymptomatic individuals.

Through monitoring viruses in wastewater, researchers are able to understand the actual prevalence of COVID-19 in the population. WBE can also be used to detect genetic variations in different regions, which means scientists can monitor the evolution of the virus over time, as well as provide valuable information on the prevalence of variant viruses. This will help to inform governments on which measures to implement, such as lockdowns and vaccinations.

There is a huge stigma surrounding the use of epidemiological tests which diagnose individuals, and therefore all data is collected based on personal information. Alternatively, wastewater-based testing can help researchers to understand epidemiology without having to access personal information.

Associate Professor Masaaki Kitajima and his team at Hokkaido University’s Division of Environmental Engineering have been working to create wastewater-based technology, which can be used to understand the prevalence of COVID-19 with lower costs and less stigma.

Professor Kitajima explained: “We are working with Shionogi, a major pharmaceutical company in Japan, to create wastewater-based technology from a combined viewpoint of academia and industry. Through this collaboration, we want to try to transfer this approach from the university to the market.

“Although we aim to operate in the business of WBE, our primary objective is to contribute to society by offering this technology to the public.”

Shionogi began work on antibiotic-resistant bacteria in wastewater three years ago, along with other pathogenic viruses. As the COVID-19 pandemic became more dangerous worldwide, the company looked to extend their research to cover COVID-19 WBE, and began to reach out to researchers who were actively working in this field in Japan.

It was here that the partnership between Associate Professor Kitajima and Shionogi started. “Shionogi researchers contacted me and I wanted to work with them because the direction of our research was agreeable,” explained Professor Kitajima.

As of April 2022, AdvanSentinel Inc. – a joint venture recently established by Shionogi and Shimadzu Corporation – is now a part of this existing collaboration between Hokkaido University and Shionogi.

Due to Japan having a relatively low number of COVID-19 patients, the concentration of the virus in wastewater is quite low.

Because of this, methods that have been successfully applied for wastewater detection of the virus in other countries have worked as the concentration of the virus is high and easy to detect. However, these methods were not sensitive enough to be used in countries such as Japan, which have lower infection rates.

To solve this issue, Associate Professor Kitajima and the Shionogi researchers developed a highly sensitive detection method, known as Efficient and Practical virus Identification System with ENhanced Sensitivity for Solids (EPISENS-S), which finds the virus in wastewater where there is a low number of infected people.2 They discovered that this method is approximately 100 times more sensitive than the polyethene glycol method, which is currently the most widely used process in Japan.

“We first examined the detectability and physical partitioning of the virus in wastewater from a quarantine hotel in Japan because we are more likely to detect the virus here. Based on the data obtained from the quarantine hotel wastewater, we then successfully developed the EPISENS-S method. We also used this wastewater to explore the applicability of WBE on facility-level wastewater,” Kitajima stated.3

EPISENS-S involves centrifuging collected wastewater samples to separate all the solids in the samples. The solids are treated with a commercially available kit to extract all the RNA, which is then reverse-transcribed and amplified to obtain a substantial amount of DNA copies.

This method is more successful than the current method, as it enables asymptomatic cases of COVID-19 to be detected and tracked. In the future, there is potential for EPISENS-S to be adapted to track other viral diseases with low infection rates and viral loads.

This technology was used in the Tokyo 2020 Olympic and Paralympic Games that were held during July and September 2021 – a time when the spread of COVID-19 was increasing rapidly. The team implemented WBE in the Olympic and Paralympic Village to ensure that the athletes and support staff were safe.

Here, daily testing of both athletes and support staff, along with sampling and testing of the wastewater in the sewage system, was carried out. The results of these tests were reported to the Tokyo 2020 Organising Committee.

Links were found between clinically reported cases and viral loads in wastewater. SARS-CoV-2 was detected in 151 wastewater samples out of the 360 samples collected from seven distinct areas of the Olympic and Paralympic Village.4 Of these 151 wastewater samples, 53 were from the Olympics and 98 were from the Paralympics, the latter being where the number of confirmed cases was higher. The strongest correlation between SARS-CoV-2 RNA load in wastewater and the presence of clinical positive areas were found in areas that had maximum viral loads in wastewater within a three-day span.5

Overall, the study showed that WBE and clinical tests can work simultaneously. By combining the two methods, COVID-19 clusters were prevented in the village. Because of this, WBE could be used to trace and control COVID-19 clusters in the future, along with use if the concentration of the virus increases.

In order to implement WBE on a large scale, it is necessary to establish a high throughput analysis system of the collected wastewater samples. Despite the development of a number of SARS-CoV-2 detection methods, there have been obstacles to their social implementation.

To overcome these obstacles, the collaborative researchers created the COPMAN method (Coagulation and Proteolysis method using Magnetic beads for the detection of Nucleic acids in wastewater) to establish an automated analysis system that can analyse a large number of samples required for the implementation of WBE at a national level.

The method is a highly sensitive detection technology, which is able to achieve rapid and stable virus recovery by using a coagulant in the virus concentration process from wastewater. As well as its ability to detect SARS-CoV-2 in wastewater, the system is expected to enable efficient detection of other viruses. Moreover, it will accelerate the social implementation of WBE, due to the fact that a large number of samples can be analysed by implementing COPMAN with humanoid robots.

The COPMAN method consists of three steps. Firstly, the concentration of viruses in wastewater using a coagulant. Next, the extraction and purification of RNA using magnetic beads, and lastly, a reverse transcription (RT)-preamplification-quantitative PCR (qPCR).

The results of the study revealed that the COPMAN method ensured the highly sensitive detection of viral RNA in wastewater. This was established in the RT-preamplification-qPCR step, where a series of enzymes exhibiting a high tolerance to PCR inhibitors derived from wastewater were employed.

Kitajima and his team compared their method with the polyethene glycol (PEG) method and the ultrafiltration method for concentration, followed by the conventional qPCR method. In comparison to the PEG precipitation method, which takes nine hours to detect the presence of the virus, the COPMAN method takes just ten minutes. Furthermore, the detection rate for the new method was 100%, which is much higher than the compared methods and demonstrates COPMAN’s high sensitivity.

Altogether, the researchers found that this method was the most efficient for detecting spikes in viruses and the highest level of pepper mild mottle virus from wastewater. COPMAN is suitable for the detection of multiple pathogens from only 10ml of wastewater in automated stations.

The traditional PEG-qPCR method mainly used liquid fractions for detection, and the EPISENS-S method, first developed by the researchers, only considers solid fractions. The COPMAN method was therefore designed to recover viruses present in both the liquid and solid fractions of wastewater using a polyaluminum chloride (PAC)-based coagulation-flocculation process.

The COPMAN method eliminates the centrifugation process as much as possible, due to its incompatibility with automation, and adopts a nucleic acid purification method using magnetic beads, more suitable for automation. This innovation makes WBE surveillance more useful and accessible and is expected to accelerate the social implementation of wide-scale WBE.

Please note, this article will also appear in the twelfth edition of our quarterly publication.

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