Predicting future outbreaks

Clover Carlisle keeps a vigilant eye on the cleanliness of Delaware’s drinking water as an Analytical Chemist and Laboratory Manager of the Environmental Lab at the Delaware Public Health Laboratory (DPHL), located in Smyrna. After 15 years in neonatal testing at DPHL, she’s spent the past six years testing samples from utilities throughout the state that provide drinking water to the public. The EPA-certified lab analyses samples for bacterial and chemical contaminants, this includes organic compounds, metals, and mercury, among other potentially dangerous elements. 

When Delaware decided to expand its water monitoring to include wastewater, the Environmental Laboratory took on the job of hunting for COVID-19 in wastewater. Wastewater-based epidemiology (WBE) is a method of monitoring disease circulation in a community in real-time. Recovering tiny fragments of SARS-CoV-2 RNA from wastewater has emerged as a new tool in the fight against COVID-19, and it has public health experts excited about its potential to detect a variety of diseases in a population before people show up at a doctor’s office with symptoms or spread contagion to others.  

While COVID-19 WBE programs have popped up around the globe, DPHL is one of the first U.S. state public health labs to adopt the strategy. “There are not a lot of other programs to reach out to for help,” Carlisle says. “There are not a whole lot of people within state public health laboratories with experience.”

Wastewater surveillance at DPHL

When the COVID-19 pandemic began, DPHL took an all-hands-on-deck approach to process the samples that were starting to flood in from all over the state by marshaling the skills—and quick trainability—of its staff. Taylor Moore, an Analytical Chemist in the Environmental Lab since 2018, switched from working as a primary analyst for disinfecting byproducts to running COVID-19 assays in the molecular lab, where she got a crash course in PCR. 

“Molecular testing was completely new to me,” Moore says. “It was a challenge at first, but it was very interesting. I got familiar with the instruments, learned how to plate, and prepare master mixes, primers, probes—all that stuff. It set me up pretty well to move into wastewater.” 

Carlisle’s interest in wastewater surveillance was piqued by the American Public Health Laboratory (APHL)’s launch of a Wastewater Surveillance Community of Practice, a forum for APHL member laboratories to share resources. 

“In these calls, it came up that QIAGEN was going to offer a pilot program in the future,” Carlisle remembers. The pilot program was for the testing of the QIAcuity digital PCR platform, a highly precise approach to sensitive and reproducible nucleic acid detection and quantification that can identify even trace amounts of fragmentary viral RNA in wastewater. Digital PCR can increase sensitivity five- to ten-fold. 

“I spoke with the DPHL Director, Christina Pleasanton, and she thought this was a good opportunity for us.”

Test pilots for dPCR

They partnered with the Delaware Department of Natural Resources and Environmental Control (DNREC), a state agency that has the authority to collect samples from wastewater treatment plants. Of the 31 publicly-owned wastewater systems in the state, DPHL receives samples from 13 of them twice a week. 

Then DPHL joined the QIAGEN pilot program in 2021. “The QIAGEN pilot program was an opportunity to be a part of a program that established a whole automated method from extraction to plating the master mix to transferring to plates,” she says. DPHL uses three QIAGEN instruments in this process. The QIAcube Connect does the extraction, the QIAgility plates the master mix and samples, and the QIAcuity runs the digital PCR experiment. Its nanoplate technology partitions the sample into 26,000 individual reactions, increasing the chances of identifying a single positive. 

Moore is one of the primary DPHL scientists operating the QIAcuity. “At first it was difficult because not a lot of public health labs knew anything about wastewater testing,” she admits. “So it was a challenge at first bringing up a new method. But the instrument is very user-friendly, and the software is very intuitive. We were trained on it and able to pick it up pretty quickly.”

Wastewater samples can be complex to work with. “As you can imagine, there's a lot of other things in wastewater samples besides SARS-CoV-2, so a lot of the issues have to do with being able to work around that, with inhibitory factors,” she says. “Some samples come with a bit more sludge than others adding to the likelihood of interfering substances.” 

Another challenge has been finding which concentration method works best for their purposes. “We were doing PEG precipitation, which goes through a centrifugation process, but it is time consuming (taking most of the day),” Moore says. “It also requires a lot of centrifuges, which took up a lot of space. We recently just switched to a different concentration method that can concentrate our samples within about four minutes. That time difference really helps quite a bit now that we're getting a lot more samples than we were at the beginning.” At first, they were processing two samples a week, but that’s grown to 26.  

From patient surveillance to WBE

“The nice thing about the QIAcuity dPCR is that it gives you a quantitative result. So we're better able to see exactly what's going on within the sample,” Moore says.

“That's what makes it such a great instrument to use,” Carlisle agrees. “It’s hard to find companies that are going to make these standards or controls to be able to report out quantitatively. Most clinical assays are designed to find out whether bacteria, virus, etc are present or not.”

She adds, “With quantitative knowledge, you can report out how many copies you have of whatever you're looking for. That's going to show you when a trend is coming. You're going to see when there are a lot of patients that are shedding the virus but are not actually showing any signs or symptoms. In theory, this is going to tell us about a week before a surge would hit.”

“Molecular testing already plays a strong role at DPHL detecting viruses and bacteria,” Carlisle says. But those samples are coming directly from the patient. Carlisle thinks WBE “may even replace patient surveillance because it's going to be able to show all these trends before patients are actually getting sick,” she says. “There's so much that can be done with wastewater. I see it growing into something that's going to be huge in the future. I think that we're going to be looking for a lot more viruses like norovirus, adenovirus, and influenza, and maybe going into looking for certain bacterias that are commonly seen in food outbreak situations, such as salmonella.”

They’ve already tested this out by looking for norovirus in wastewater. “We were able to extract and use a primer probe set that’s very similar to what they would use for patient analysis in the molecular lab. We were able to get good results from that. So we know that we're able to do it for wastewater. Now we’re just waiting to see if the assay kits will become available for us in the future.”