Can Biobot also test samples from effluent or sludge after the primary clarifier?
At this time, we do not test effluent water or sludge as these do not contain useful information for Biobot’s mission of leveraging wastewater for public health. Our lab instead focuses on influent wastewater, including samples collected after the grit removal and bar screen processes. Our methods are best tailored for raw influent samples––the dirtier the better!
At what frequency should we be testing our wastewater to get actionable data on trends in my community?
The optimal sampling strategy for your community, whether a municipality or a building environment, depends on how you intend to use the data. We generally recommend sampling at least weekly. If you plan to use wastewater as an early warning system, you may need to sample more often than weekly so that trends based on multiple samples can be quickly evaluated.
How many people do I need to have in my catchment to detect a case?
Our current protocol has a limit of detection (LOD) of 3,600 SARS-CoV-2 virus copies / Liter (3.6 copies/mL) of sewage. We reliably (>99%) detect the virus when there is at least 1 infected person in a population of 6,500 people. In other words, our methods are sensitive enough to detect the virus even if there is only 1 infected person in a community of 6,500 people or smaller.
Can you tell if the SARS-CoV-2 virus is alive or infectious?
No. qPCR does not tell us whether the virus is dead or alive, it just detects fragments of the genetic material of the virus.
Can people catch the virus from handling wastewater?
There is no evidence to date that anyone has become sick with COVID-19 because of exposure from wastewater. Standard practices associated with wastewater treatment plant operations should be sufficient to protect wastewater workers from SARS-CoV-2. These standard practices can include engineering and administrative controls, handwashing, specific safe work practices, and personal protective equipment normally required when handling untreated wastewater. Beyond CDC recommendations for how to protect against COVID-19, no additional COVID-19-specific protections are recommended for workers managing wastewater, including those at wastewater treatment facilities.
How do you quantify the SARS-CoV-2 virus in samples?
We quantify the concentration of the virus through a method called quantitative polymerase chain reaction (qPCR), which is adapted from the CDC protocols for clinical diagnostics. qPCR looks for a genetic signature of SARS-CoV-2, the virus that causes COVID-19. The concentration of the virus is indicated in the output of qPCR, called Ct values. We used the primers and probes designed by the CDC for qPCR analysis. Unlike clinical specimens, viruses are very diluted in wastewater, so we apply sample preparations to detect the virus with high sensitivity.
Do Biobot methods measure the N1 or N2 nucleocapsid gene?
Yes, we measure both N1 and N2 nucleocapsid genes to improve the specificity of the analysis. We take the average of the N1 and N2 results and report that single number as the raw virus concentration. We also adjust that average concentration to PMMV, which is reported as the normalized concentration.
How is a normalized viral concentration derived?
We normalize the SARS-CoV-2 viral concentration to a fecal indicator to account for differences in dilution or fecal content between samples. We use the pepper mild mottle virus (PMMV) as this fecal indicator, which is an RNA virus that is commonly excreted in the stool.
The normalized virus concentration value is derived by adjusting the raw virus concentration to the level of the PMMV fecal marker in each sample relative to a reference value of PMMV.
Normalizing the data to a virus universally found in stool is the best way to control for dilution and variability in sampling (i.e. how much stool is actually in each sample). If your samples tend to be more dilute than the average sample in our dataset, you’ll see consistently higher normalized values than your raw concentration. That is because the dilution in your samples will lead to lower PMMV than average, and so your sample concentrations will be corrected upwards in the normalization process.
How do you address the issue of dilution? (leaky pipes, rain, snow)
We address the issue of dilution by normalizing the SARS-CoV-2 virus concentration to the concentration of a fecal biomarker.
For example, if wastewater is diluted by 20% due to rain, the SARS-CoV-2 concentration will be reduced by 20%, but so will the PMMV concentration. As a result, the SARS-CoV-2 concentration normalized to PMMV remains unaffected by dilution.
Why do we need a composite sample?
The composite sampler provides us a reliable look into the health of the whole population contributing to the waste stream. If we were to test one sample taken at one instance in time (i.e. a grab sample), that sample would only capture a small subset of the overall population that contributes to the sampling catchment. A composite sampler smooths out the variability introduced by sampling only a subset of the entire wastewater stream.
How frequently should we pump wastewater during 24-hour composite sampling?
Pumping frequency depends on population size, but in general, we recommend a pumping frequency of every fifteen minutes for municipal customers.
Does data from wastewater only reflect people who currently have COVID-19 or does it also include those who have fully recovered?
Our results represent people who are actively shedding the virus in their stool. Current research indicates that most viral shedding in stool takes place in the first few days after infection. Therefore, wastewater likely primarily represents people who have been recently infected with COVID-19, even if they have not yet developed symptoms. It does not include those who are fully recovered and no longer shedding the virus. In epidemiology terms, our data from wastewater is indicative of disease incidence. You can read more about this in our paper.
Where can I find literature supporting Biobot’s method?
Here are four references for our work. Downloadable PDFs are available from all four links. Biobot staff continue to publish cutting-edge academic papers––you can follow @BiobotAnalytics on Twitter to stay in touch with our most up-to-date work. For more publications by the Biobot team and our collaborators, please check https://biobot.io/science/
1) SARS-CoV-2 titers in wastewater are higher than expected from clinically confirmed cases
Our team describes the first successful detection and quantification of SARS-CoV-2 in wastewater samples collected in the US (in Massachusetts). We validate our qPCR assay by sequencing the PCR product and matching it to SARS-CoV-2, and we present our thought process to estimate the number of COVID-19 infections based on wastewater titers. We also report that wastewater indicates a higher level of community infection than reported clinical cases.
2) SARS-CoV-2 titers in wastewater foreshadow dynamics and clinical presentation of new COVID-19 cases
Our team describes a time series analysis of samples collected from January to May in Massachusetts showing that we detected SARS-CoV-2 in wastewater days before the first reported clinical cases in March. We also present evidence that wastewater is a leading indicator for new COVID-19 cases (incidence) 4-10 days in advance. Wastewater provides this early warning system because most SARS-CoV-2 virus is shed in stool in the first three days of the infection, before symptoms appear and therefore before people get diagnosed with a clinical test. It is important to notice that based on our work, wastewater better reflects COVID-19 incidence rather than prevalence.
3) Presence of SARS-Coronavirus-2 RNA in sewage and correlation with reported COVID-19 prevalence in the early stage of the epidemic in The Netherlands
Peer-reviewed article published by a wastewater-epidemiology research team in The Netherlands where they detected SARS-CoV-2 in wastewater days before the first reported clinical cases in their country, further supporting that wastewater provides an early warning for COVID-19 outbreaks.
4) First confirmed detection of SARS-CoV-2 in untreated wastewater in Australia: A proof of concept for the wastewater surveillance of COVID-19 in the community
Peer-reviewed article published by a wastewater-epidemiology research team in Australia. They also validate their qPCR assay through sequencing of the PCR product, and present methods to estimate the number of COVID-19 infections based on wastewater titers.