How do you measure 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 use 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, a fecal indicator, in order to account for dilution due to flow rates or precipitation, and with an adjustment factor to account for changes in lab protocol.
How does Biobot normalize for inflow and infiltration?
We normalize the SARS-CoV-2 viral concentration to a fecal strength 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 stool.
The effective 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. Then, we use an adjustment factor to account for other changes in lab protocol.
Normalizing the data to a virus universally found in stool is a good 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 effective concentration values than your raw concentration. That is because the dilution in your samples will lead to lower PMMoV than average, and so your sample concentrations will be corrected upwards in the effective concentration calculation.
For example, if wastewater is diluted by 20% due to rain, the SARS-CoV-2 raw concentration will be reduced by 20%, but so will the PMMV concentration. As a result, the effective concentration remains unaffected by dilution.
Please reference our Effective Concentration FAQ for more information.
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 9,000 SARS-CoV-2 virus copies / Liter (9.0 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.
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 begins. 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 see 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.