The wastewater project is led by Dr Joanne Hewitt, head of ESR's Environmental Virology Laboratory and ESR Science Leader, Dr Brent Gilpin. Initial work on the project has been funded by the MBIE’s Covid-19 Innovation Acceleration Fund, with the Ministry of Health now funding further application of the work as part of New Zealand’s COVID-19 response.
How do you collect it and test it?
The preferred option is the automatic composite sample. This is where a pump automatically collects a small volume of wastewater every 15 minutes over 24 hours. These are already setup at most wastewater treatment plants and most of the samples are taken this way. It means you get a sample which is reflective of multiple points in time over the day. While many treatment plants have these samplers, some don’t and if you are looking to investigate a smaller area, sampling from a manhole for instance, you have to use grab sampling, which is exactly as it sounds. You are taking a sample directly from the wastewater at one point in time. Which means you basically get a snapshot in time which is dependent on diffusions and flows, so it’s less reflective of the overall virus in the sewage for that time.
However, ESR are looking at different approaches, such as using passive samplers that can capture viruses from wastewater as it passes through it. These could be rapidly deployed at locations across the sewage network when composite samplers are not available, like the extra sites sampled for the latest investigation.
Once we receive the one litre samples at ESR’s laboratory in Wellington, our laboratory staff have become very good at processing them quickly. First, we take up to 500 ml from those samples and store the rest. As viruses can be attached to the solids (poo) and be in the water, we use methods that will recover viruses from both parts, so we don’t lose any virus. This is important if we don't expect a high level of virus in the sample. Then we concentrate it all down to about half a teaspoon, basically removing all the water and leaving the virus behind. From there it’s the same process as you go through for regular testing for the virus of someone’s swab, we extract the viruses, turn the RNA into DNA and run it on a PCR to tell us if the sample is positive or negative for the virus.
We also add a cat coronavirus and a mouse norovirus to every sample we test as a control to see how good our methods are. The cat coronavirus is a good control for SARS CoV-2, while the murine norovirus is more like the enteric viruses we usually look for in wastewater. We store the other 500 ml to confirm an unexpected result. Remaining viral nucleic acid and concentrated samples are also being stored for future work on human viruses in wastewater.
Can we trust the results?
There are two factors – specificity and sensitivity. How do we know we’ve detected what we want to detect and how sensitive is the test at detecting it. We have been working for many months of both as part of the ongoing research.
In terms of specificity – we are very confident in the precision. When we detect viral RNA in a sample, we are very confident that it is actually from SARS-CoV-2. PCR is a widely used methodology, is extremely sensitive and can produce false positive results particularly when we are looking for things at very low levels. This is something we constantly work to guard against. We have a lot of positive and negative controls, and lots of replicates. Before reporting a positive, particularly a very low level unexpected positive, we would always repeat the PCR, and then the whole extraction on a stored replicate sample. It’s tough on the lab as this would invariably require working late into the night, but we are committed to results that we are completely confident in, and which can inform decisions made by Government.
The second part of this is sensitivity. It is theoretically possible to detect a single person shedding virus, but unlikely in practice – at least in a reproducible manner. Based on what we have observed so far, our test is sensitive enough to discover approximately 10 cases in an area of 100,000 people. That’s not a hard and fast rule as some people shed at different rates based on time of infection, and there are many other factors that affect detection. Generally, we would need more cases in large catchments to increase the likelihood of detection because as more people contribute to a sewerage stream there will be dilution in the wastewater which will alter the level of sensitivity.
It’s kind of like sharks detecting blood in the water. If we think of the blood as the virus and the sharks as our sampling. People say that sharks can detect a single drop of blood from hundreds of metres away. The more drops of blood in the water, the more likely they detect it. On the same line, the more sharks in the water in the right area, the higher likelihood they detect it.
What are the limitations to it - what can't it tell us ?
Wastewater testing can tell us someone in the network shedding virus, but not specifically where or who is shedding the virus, or the exact number of people who have contributed to a positive detection. Shedding levels can vary significantly between individuals and at different stages in an infection and not every infected person may shed the virus.
People who have been infected may continue to shed virus after they are no longer infectious. Published data suggests that this can continue for weeks after the initial illness. However, most of the data we have in this space comes from hospitalised individuals, most of whom are very sick and some with ongoing immune issues. Not really the situation we have in New Zealand with those in managed isolation, where a third of cases are asymptomatic. Also, most are younger and fortunately most have milder symptoms. Therefore, how long shedding in faeces occurs in most of the COVID-19 cases in New Zealand is an unknown. What we do know is that this post infectious shedding by people will produce much lower levels of virus in faeces, which is good for New Zealand’s unique situation with the small number of cases we currently have. We have tested now several hundred wastewater samples, many of which will have had people who previously had COVID-19 contributing to them. What we see when an outbreak occurs is a consistent detection of virus at relatively high levels. That’s one of the reasons we like to collect a series of samples. Historical cases could produce the occasional sporadic low-level result, but they are not going to produce ongoing detectable levels.
The protocol we developed with the Ministry of Health is that if have a very low-level unexpected detection, to immediately analysis a second sample taken at the same time, and then a repeat sample taken a day or two after.
It is also important to remember that not everyone’s home is connected to a municipal wastewater system. Many of these people may however work or socialise in places that are connected to sewer and therefore still get captured by wastewater testing. And those in most isolated places are less likely to get infected, and less likely to spread the virus to others, than those living in higher populated centres.
There are always complicating factors, but we continue to learn and build upon our findings.
How is the research addressing this?
The ESR team have learnt a lot by undertaking daily sampling of wastewater from the Jet Park Hotel since July 2020, and from the South Western Interceptor, where the Jet Park wastewater mixes with 130,000 other Aucklanders. The Jet Park wastewater is almost always positive for SARS-CoV-2, and this allows ESR to better understand detections in municipal wastewater.
It's a controlled place to study detection because we know how many positives are inside Jet Park. So, when we see the positive detection right outside Jet Park, we can match it with the number of positive guests. Then we check whether we get that positive further down the line at the South Western Interceptor where it mixes with more wastewater. We have found this municipal treatment plant is positive for SARS-CoV-2 approximately 50 per cent of the time, but that varies, and detections correlate with higher numbers of infectious cases in the Jet Park hotel. We have also had detections in wastewater from Christchurch and Rotorua which we could match with known cases in
We use this knowledge and apply it to the other sites not showing any positives.
Who is ESR working with on this?
ESR is part of ColoSSoS (Collaboration on Sewage Surveillance of SARS-CoV-2)(external link) a project led by Water Research Australia and operating in parts of Australia, New Zealand and the Mekong countries. The project has the goal of ensuring experts are liaising and using the most reliable and robust methods and techniques to find a way to conduct this work for the good of communities.
We also have to remember, that this research is going to be useful for other viruses longer-term. The whole area of wastewater epidemiology is a burgeoning area worldwide. Illicit drugs and infectious viruses are just two of the possible areas that wastewater analysis may allow us to understand. The smart toilet of the future may analyse what you deposit in your toilet to help with your personal wellbeing and health, while on a societal level wastewater analysis may allow us to better understand the behaviours and health of New Zealanders across a whole range of health and wellbeing issues. You might say there really is “information gold” in wastewater, and wastewater epidemiology is something you are only going to see more of in the coming years.