SARS-CoV-2 RNA is enriched in the solids fraction of wastewater

By Jocelyn Solis-MoreiraNov 14 2021

New research led by Alexandria Boehm of Stanford University in California shows that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ribonucleic acid (RNA) in wastewater can be detected by measuring solid samples and flowing sewage water samples.

SARS-CoV-2 RNA tends to cluster in small masses of solids, making direct extraction of nucleic acids possible when there are low viral RNA levels.

Both solid and liquid methods were sensitive enough to detect low levels of SARS-CoV-2 RNA. Unless there are high COVID-19 caseloads in the surrounding community, the researchers note that wastewater should be processed beforehand if there are low concentrations of SARS-CoV-2 RNA in large volumes.

The study “SARS-CoV-2 RNA is enriched by orders of magnitude in solid relative to liquid wastewater at publicly owned treatment works” was recently published on the medRxiv* preprint server.

Study: SARS-CoV-2 RNA is enriched by orders of magnitude in solid relative to liquid wastewater at publicly owned treatment works. Image Credit: APChanel / Shutterstock

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources

Study details

The researchers measured the amount of SARS-CoV-2 RNA flowing in solid and raw sewage water from five publicly owned treatment works.

SARS-CoV-2 RNA levels were higher in solids by almost three to four orders of magnitude.

Previous research looking at bacteria and viruses in wastewater also showed a similar affinity towards wastewater solids. Similarly, the current study showed that other viruses such as the Pepper Mild Mottle Virus (PMMoV) are abundant in solid fractions rather than dispersed in flowing wastewater.

The solids that entered the public sewages were suspended in the water and later made into sludge. The researchers estimate that the concentration of SARS-CoV-2 RNA in the solids is based on the concentration in suspended solids and that solids have three orders of magnitude more SARS-CoV-2 RNA than flowing wastewater. Based on the assumptions, they estimate only 10% of SARS-CoV-2 RNA is in flowing wastewater.

Solid approach requires less sample to measure SARS-CoV-2 RNA

The ratio of concentration in both solids and liquid wastewater can be measured using the partitioning coefficient K_d. To determine the characteristics of solids in wastewater, the researchers next measured K_d to PMMoV in solids.

PMMoV was selected to measure fecal strength and as a measure of organic substances in the solids.

K_d is positively correlated with PMMoV concentration in solids.

Based on the results, there is a possibility that K_d may be altered by how SARS-CoV-2 RNA is being measured. Solid approaches are similar to pre-analysis and extraction methods because RNA is concentrated in a small volume of the sample of less than 1 milliliter. For liquid approaches, there is a need for diverse pre-analytical and a series of extraction steps requiring a collection and concentration of SARS-CoV-2 RNA from over 20 milliliters of liquid.

As a result, liquid approaches may make K_d lower when measuring SARS-CoV-2 RNA levels in flowing wastewater.

Time series of (top to bottom) SARS-CoV-2 targets N1 or N measured in solids (cp/g dry weight), concentration measured in influent (cp/mL), and laboratory-confirmed SARS-CoV-2 incidence rate for each of the five POTWs over their respective duration of sample collection. N was measured for OS solids and N1 for all other data sets. Each wastewater data point represents SARS-CoV-2 RNA concentration for a single sample as reported by the respective laboratory. Replication was performed differently for each lab (see SI). Samples above the lower measurement limit are shown as filled circles. Samples that resulted in ND, shown as empty circles, were substituted with a value half of the sample’s lower measurement limit. Lines for solids and influent are locally weighted scatterplot smoothing (lowess) with value of α that minimizes the residual for each dataset . Lines for clinical are 7-day centered smoothed averages

Detection sensitivity of SARS-CoV-2 RNA in wastewater

The researchers next looked at the sensitivity of each approach at detecting SARS-CoV-2 when COVID-19 cases were low in the area. The low COVID-19 incidence rate should have yielded non-detectable levels of viral RNA.

Results showed both solid and liquid methods were sensitive in detecting viral RNA across all public sewages.

Their detection rate for SARS-CoV-2 RNA fell between ~1 and ~10/100,000 and was most sensitive when they could detect a <1/100,000 incidence rate.

Though the researchers note that it is still unclear how sensitive detection methods in wastewater need to be to provide an informative pandemic response. The results suggest both methods could identify about 1 to 100 cases of COVID-19.

Overall, both methods were representative and sensitive for SARS-CoV-2 RNA detection. Solid detection can also be used with pre-analytical methods and scalable.

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources