Wastewater surveillance has become an important tool in the effort to stop the spread of COVID-19. Monitoring the presence of SARS-CoV-2, the virus that causes COVID-19, in wastewater samples can provide an early warning sign of COVID-19 outbreaks in a population.

A wastewater surveillance method developed at the University of Missouri not only detects the presence of SARS-CoV-2 but also quantitates the abundance of the virus in wastewater, allowing scientists to determine the degree of infection in a population. We recently sat down with Dr. Chung-Ho Lin to discuss his method and how Gilson PIPETMAX® was incorporated into their workflow, and how this approach impacts efforts to mitigate the spread of COVID-19.

Customer Profile: Dr. Chung-Ho Lin, University of Missouri

Dr. Lin is a research associate professor at the University of Missouri and is the lead scientist in charge of the bioremediation program at the MU Center for Agroforestry in the College of Agriculture, Food, and Natural Resources.

His focus is on bioremediation chemistry, including method development for the detection and quantification of molecular genetic materials and pollutants in the environment; isolating, identifying, and quantifying bioactive compounds in plant material; and exploring industrial applications for wastewater.

 

Wastewater Surveillance Program

Lin is a research associate professor at the University of Missouri and is the lead scientist in charge of the bioremediation program at the MU Center for Agroforestry. Having been tasked to develop methods to quantify SARS-CoV-2 and its variants in wastewater last May, Lin and Dr. Marc Johnson at the University of Missouri, in collaboration with Missouri state agencies,developed the largest SARS-CoV-2 wastewater surveillance program in the US. “We have been keeping a close eye on the copy number of SARS-CoV-2 and its variants in wastewater across almost 100 facilities in the state of Missouri, including the municipal wastewater treatment plants, nursing homes, military bases, training camps, and correctional facilities,” says Lin. “It allows us to provide an early warning of the presence of SARS-CoV-2, and also to evaluate mitigation efforts across the state.”

Absolute Quantitative RT-qPCR Testing to Determine Infection Spread

Their method utilizes absolute quantitative real-time PCR (RT-qPCR) to quantify SARS-CoV-2 copy numbers in wastewater. To perform testing, they extract RNA from wastewater samples, perform RT-qPCR, and then compare the samples to a calibration curve to determine viral copy numbers. Using this method, they can quantify down to as few as five copies of the virus in an RNA sample extract and predict the degree of infection in a population.

As Lin explains, “The quantitative data allows us to evaluate how many people are infected. A lot of people are infected but areasymptomatic. They don’t know that they are sick and don’t have any symptoms, but they are still spreading the virus. This type of surveillance testing allows us to get an overall picture of the scope and the trend of infection over time.”

Advantages for Congregate Settings

This approach is cost-effective, especially for congregate settings such as nursing homes and correctional facilities. Accordingto Lin, “Our surveillance project covers about 70% of the Missouri population, the equivalent of 3.7 million people. If you split the cost, it’s only one cent per person, as opposed to an individual clinical swab test, which can cost $200 or more.”

Wastewater surveillance also provides an early indication of infection in a population, which can help prevent outbreaks. “We can typically give facilities seven days warning time once we’ve identified the presence of SARS-CoV-2. This allows them to decide how to allocate clinical testing resources more efficiently because it’s expensive to test everyone individually. Once they see the spike in the virus, they can immediately implement mitigation efforts and try to minimize the chance of an outbreak. In our experience, we have prevented several major outbreaks in corrections facilities across the state,” says Lin.

PIPETMAX®

Lin has incorporated our automated liquid handler, PIPETMAX, into their testing workflow to prepare samples for RT-qPCR analysis. PIPETMAX is a compact, benchtop-sized automated pipetting robot used for the efficient processing of high-throughput assays. Using PIPETMAN technology, the robot transfers liquids with high accuracy and precision, helping to eliminate user-to-user variability.

Lin has used Gilson automated liquid handling systems in the past. “I always have a positive experience using Gilson products. I have found them to be very robust and reliable. That’s why I first looked to Gilson when I needed an automated system to see what they had to offer that I could take advantage of for RT-qPCR sample preparation.”

The Importance of Automation

Having an automated liquid handling system has been an essential component of their protocol. “Our lab works 13-14 hours per day to make sure we can have our data reported quickly, sometimes within 12 hours. Automation is a very critical component to allow us to have this kind of productivity and enable us to inform the general public quickly about any health risks,” says Lin.

Every week they process approximately 2,000 RT-qPCR reactions to quantify the SARS-CoV-2 and its variants in 200 wastewater samples using PIPETMAX. They expect to double that capacity within a few weeks. “It’s running almost non-stop. Imagine how many people it would take to carry out this kind of workload and offer this consistency, precision, and accuracy. Without PIPETMAX, it’s impossible,” says Lin.

PIPETMAX has not only helped with their sample processing capacity but has also helped them eliminate pipetting variability and have confidence in their results. “With 4-5 μL pipetting volumes, getting that precision and accuracy is impossible manually. Having an automated system gives us peace of mind. It also allows us to stay on schedule because we know how long it takes for the system to load the samples. If we do everything manually, there are a lot of uncertainties. Different people may have different habits that introduce variation. Having a robot allows us to operate more efficiently and also ensures we have the quality assurance and quality of analysis we would like to have,” says Lin.

Tracking Variants

Recently, the team has shifted half of its effort from routine surveillance to tracking the UK, Brazilian, and South African SARS-CoV-2 variants. “Based on our wastewater surveillance, we learned that over the past eight weeks, we went from having 0% variants to almost 80-95% variants,” explains Lin.

Compared to individual diagnostic tests, the wastewater surveillance approach generates a population pool that can be rapidly sequenced at a low cost to monitor for SARS-CoV-2 variants, providing results in time to be useful in mitigation efforts. According to Lin, in the clinical approach, if someone is suspected of being infected with a SARS-CoV-2 variant, a nasal swab test would be performed and sent to the Centers for Disease Control (CDC) for genomic sequencing. “It will take at least three weeks until they learn anything. It is almost impossible to figure out the scope of the variant infections through the clinical approach,” he says.

Wastewater surveillance also generates more information compared to clinical tracking of variants. “Approximately four weeks ago, we learned that SARS-CoV-2 variants were present in almost 24% of the wastewater samples we tested. But in terms of clinical tracking at the state level, we had only identified one patient that was infected with a variant.”

They are also closely tracking variants to help inform mitigation efforts. “The current massive vaccination plan is basically applying selection pressure to the virus. The virus wants to survive, so it keeps mutating,” says Lin. “Most of the current treatment approaches were designed for the original strain. Having more information on the variants can help determine whether those treatment options will still be effective and help our mitigation efforts.”

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A Collaborative Effort

The University of MO program is part of the CDC wastewater surveillance consortium. According to Lin, every state is interested in the wastewater surveillance approach. “We’ve been on monthly CDC consortium calls, and there were approximately 150-250 scientists from across the states. Right now, I think every state has something similar to this,” says Lin.

They also share their information with colleagues through the CDC and share their methods with non-profit organizations such as the Water Research Foundation and the National Hygiene Laboratory.

Looking Ahead

Lin expects wastewater surveillance to grow in importance as a tool to help control the spread of COVID-19. “I can see within a couple of months when the majority of the population is vaccinated; clinical testing will be reduced significantly. Then I think wastewater testing will become more important,” says Lin.

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