Exploring Wastewater Surveillance for SARS-CoV-2 with Dr. Sarah Philo
Wastewater-based epidemiology (WBE) has been around for a while and has been used to track drugs of abuse, chemical waste, and pathogens alike. It may not be the most glorious of samples to work with, but wastewater has proven to be a valuable way to do community-wide monitoring. The COVID-19 pandemic brought new attention and focus to WBE once it was shown it could be used to detect the SARS-CoV-2 virus to alert public health officials to outbreaks, often before clinical symptoms presented.
Wastewater surveillance with Dr. Sarah Philo
Our “gene-ius” guest is Dr. Sarah Philo, a postdoctoral research associate with the Wastewater Surveillance for SARS-CoV-2 and Emerging Public Health Threats Research Coordination Network at the University of Notre Dame. In our conversation, Sarah talks about how she found this area of research and the “joys” of working with wastewater samples. We learn about how she and her team have used both qPCR and dPCR to detect and quantify SARS-CoV-2 and antimicrobial resistance genes in wastewater.
The conversation also touches on several other interesting and informative topics that include a “one health” approach to public health, the importance of teamwork in academics, considerations when selecting a graduate research program, the importance of passion in science, and how science is the “punk rock” discipline within academia.
To learn more, listen to the Absolute Gene-ius dPCR Podcast Episode at www.thermofisher.com/absolutegeneius
Transcript from Absolute Gene-ius episode “Tales of a pandemic PhD.”
An introduction to wastewater surveillance
Interviewer: At this point, a lot of people have definitely heard about the SARS-CoV-2 virus, but I feel like there’s still some room to learn about what wastewater surveillance is, what that looks like. Can you give us a little bit of background? What do you do? What does that world look like?
Dr. Sarah Philo: What the world looks like, that is a huge, huge question. I mean, people are, historically have been looking for things like poliovirus in wastewater for decades. Now, it’s sort of transitioning into a lot of different infectious diseases. People have been looking for, like drug residues for a while. So, it’s just there’s a lot of different things that you could do with wastewater, and what you can look for.
Interviewer: Describe that process a little bit. So wastewater, you know, it’s a collection of sewage. What does it look like?
Dr. Sarah Philo: Maybe I won’t say exactly what it looks like, that might not be appropriate for soft stomachs. There’s a couple of different places that you can collect samples. So, one thing that was implemented a lot during the pandemic was actually collecting either at like dorm effluent on college campuses. So the pipe that comes right out of a dorm, or from like a manhole on campus that collected from a few dorms, and that would give you an idea of like a smaller community. And that was for colleges that were doing a lot of like regular testing of their students. And then they could actually isolate students who are sick. You can also collect at like, the effluent from hospitals. So if you’re looking for what’s going on in that hospital. A lot of the work that I did in my dissertation was actually collecting at the beginning of the wastewater treatment plant, or the influent as we call it, and that gave us an idea of like the entire wastewater catchment area, everything that is collected by that treatment plant.
Interviewer: I hardly know anything about this. So this might be a very ignorant question. But you guys literally you just like somebody goes in there, and they just they scoop a sample. And there it is. That’s, I mean…
Dr. Sarah Philo: So a lot of my samples were literally just like somebody took a Nalgene bottle, like, you know, the liter size water bottles, dip a bucket, and then you fill up that bottle. And that’s called like a ‘grab sample’ where it’s just like a one time you just grab it, you go. There are also things called autosamplers, which it’s a machine that you hook up at the pipe level. Every 15 minutes, it will collect some of the wastewater and you can do that over the course of 24 hours, 12 hours, so it will give you like a composite sample of all the wastewater over the course of 24 hours usually is what those are.
Research and detection methods
Interviewer: So, what happens after you get that sample? Let’s say you’ve detected some levels of SARS-CoV-2 in the wastewater. Has your research led to maybe some policy implementation or, how does it help guide you know what’s going on out there?
Dr. Sarah Philo: I wish I could say that my research directly guided policy, you know, that’s what we hope for. But my work definitely focused more on the research side. It all sort of comes back to this use case question of like, what is the goal? Early on in the pandemic, there was a lot of lofty goals, of thinking oh, we can estimate the number of people who are sick. That’s pretty difficult, depending on which catchment area you’re looking at. So a lot of the work now has transitioned to looking at trends. So, is SARS-CoV-2 rising or falling, or is it staying stable, and then also looking for those variants. And then there’s a lot of push now also to look at, you know, presence absence in long term care facilities. So like, if you have a retirement home, you could test the wastewater there. It really sort of depends on like, what the goal is of what actually happens with that data?
Interviewer: How do you go about detecting the virus? What did the methods look like there?
Dr. Sarah Philo: So there were people were doing various forms of digital PCR, which tends to be less affected by environmental inhibitors. We decided to keep with the qPCR just because it’s what we had done the whole time, and then we could have like this big cohesive dataset, knowing that it probably wasn’t as sensitive as something like a digital PCR would be. Some people are trying to work with different like rapid PCR protocols. So there’s a lot of stuff that has been developed and is being developed right now for SARS-CoV-2 and wastewater specifically, but most of the work I would say, is qPCR, and then various forms of digital PCR. And then there’s a lot of sequencing that goes on top of that, to figure out what variants might be there.
PCR in wastewater research
Interviewer: Going into the methods a little bit more you mentioned, you know, differences of qPCR, and digital PCR. Did you utilize digital PCR? And what did that look like for your research?
Dr. Sarah Philo: We didn’t get a digital PCR system until about halfway through some of the work. So a lot of the digital work that I did with SARS was involved with like comparing detection between qPCR and dPCR. And then we did, whenever we would get a new positive control, we would quantify that on the digital PCR just so that we knew what it was like instead of just going by whatever the lot number was that they gave us. Just to make sure that those numbers lined up and everything.
Interviewer: Based on your knowledge, or what else is going on in your lab, are there other ways you’re utilizing digital PCR in terms of SARS-CoV-2 surveillance? Or in other organisms, are you looking at possible other organisms to, you know, take a look at, and try to bring up surveillance programs for those?
Dr. Sarah Philo: That’s the big question right now with wastewater surveillance is like, how do we take all of this expanded capacity and interest beyond to the next thing, right? Like SARS-CoV-2 is basically everywhere at this point. But how can we use a lot of this to look for other things. And one of the big pushes has been to look for like antimicrobial resistance in bacteria. You know that has been a problem for medicine for a while, and it’s only going to become a bigger problem. I did another piece of my dissertation looking at antimicrobial resistance. One piece was looking at if those resistance genes increased, along with the first, like late 2020 COVID peak. You know doctors prescribed antibiotics because they didn’t have a lot else at that point for COVID. And for people who are hospitalized, it was to stop them from getting secondary infections. So we were asking, do any resistance genes increase along with that time period? And then I also did a little bit of a comparison between digital PCR and qPCR.
Interviewer: What did some of your results show when you actually were to compare those methods?
Dr. Sarah Philo: Like I mentioned, we tested for some different antimicrobial resistance genes with qPCR and dPCR. And pretty much all of them had a higher quantification with dPCR than with qPCR. Which makes sense, at least, knowing all of the inhibitors in wastewater and environmental samples tend to make qPCR a little bit less efficient. The one thing that I would say for anyone working with environmental samples is that I found it was really helpful to like test them in qPCR first to kind of get a general idea of how much was there. Because with digital PCR it can be very easy to overload the compartments or dilute it past beyond what can be detected. So just kind of having an idea of like, roughly how much is in your sample before, whether you do like one every 10 or whatever. So I found that was really helpful actually knowing like, okay, I think there’s roughly this many gene copies in this sample, I’m going to dilute it out this much. So that it should be within that like band of like optimizing the digital PCR.
Interviewer: Were you able to actually, you know, determine maybe a little bit more precisely the gene copies that were there using digital PCR over qPCR?
Dr. Sarah Philo: Yes, I didn’t do a lot of like, variability testing, I guess. It’s like what is the standard deviation around these. I will say that the gene copies that we detected in digital PCR were higher than what we detected with qPCR. So this is a big problem with wastewater, it’s just like, when you extract everything, a lot of times the inhibitors are also still present. Just because there’s a lot of things in wastewater that can be kind of hard to clean up. It’s pretty well established that like, PCR and qPCR do have some effects from those inhibitors. But with digital PCR that tends to be less of an issue.
Interviewer: Where do you see the future of wastewater epidemiology, when it comes to digital PCR and implementing digital PCR as a as a method for different surveillance programs?
Dr. Sarah Philo: Wastewater surveillance is talking about its future and like where it’s trying to go. But I think digital PCR has a really good place at having higher efficiency of those reactions. So a better idea of like, what is actually here, and how many numbers are here, compared to qPCR. And I think it could really help with finding some of those more rare targets. On like a presence absence basis, because if something in your wastewater is at the limit of detection of your qPCR assay, and then you throw in all of that inhibition on top of it, it’s just going to be a lot harder to find. Whereas if, with digital PCR, if you get rid of some of those inhibition effects, you’re probably more likely to find a lot of those rare targets than you would be otherwise.