Testing settled solids in sewage for SARS-CoV-2 more sensitive approach than testing influent

Many studies on the containment of the ongoing COVID-19 pandemic have dealt with the possible role of wastewater in predicting and detecting future outbreaks and understanding the epidemiology of COVID-19 disease. A recent study published on the preprint server medRxiv* in September 2020 shows that solid waste in sewage is a better substrate for testing than influent.

The interest in wastewater has been promoted by the shortage of diagnostic kits, the high percentage of asymptomatic and mild cases that are never identified, and the long lag in reporting test results.

Reflecting on the importance of wastewater testing, the United States Center for Disease Control (CDC) set up the National Wastewater Surveillance System in order to use epidemiological data derived from this area to shape COVID-19 responses.  Many other organizations are doing the same.

Wastewater Influent

Several studies aimed to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater influent, first concentrating the viral particles by filtration or flocculation followed by polymerase chain reaction for viral RNA. However, earlier experiments suggest that wastewater solids may have 1,000-fold higher concentrations of the virus relative to the influent, and should be monitored for greater detection sensitivity.

Two-Phase Study

In the current study, the researchers from Stanford University, LAC National Accelerator Laboratory, Monterey Bay Aquarium Research Institute, University of San Francisco, County of Santa Clara Public Health Department, and the University of Michigan first carried out a direct comparison of SARS-CoV-2 concentrations in wastewater influent and settled solids obtained from two wastewater treatment plants over about a week while cases were still rising in the early part of the pandemic and using different methods of analysis.

This was followed by the use of more refined methods to study samples of primary settled solids collected almost daily over ~90 days from a wastewater treatment plant, to understand how this correlates to the COVID-19 case trajectory.

The researchers were able to assess the amounts of the N1, and N2 viral targets in feces, as well as how much of the virus could be recovered from wastewater and the concentration of feces in wastewater. For the latter two objectives, they tracked the amount of bovine coronavirus (BCoV) and pepper mild mottled virus (PMMoV), respectively.

The researchers say, “Although the work focuses on a single pandemic virus, the results will be relevant for a wide suite of viral targets that have an affinity for solids.”

RT-QPCR vs. ddPCR Assays

The researchers compared viral detection by quantitative reverse transcription-polymerase chain reaction (RT PCR) and digital droplet (dd) RT-PCR, the latter done in one or two steps. They found that qPCR was ~80% to 105% efficient. RT PCR found targets in one of five samples and 4 of 7 samples from settled solids from plant A and plant B, respectively.

Using ddRT PCR, the viral targets were detected in none of plant A and all 7 from plant B. These results were also consistent across the biological replicates compared to qRT PCR. The performance of the latter was probably better because of the higher occurrence of viral RNA in the ddRT PCR samples, or the absence of viral inhibition.

Assay Inhibition

The researchers then examined RT PCR inhibition using endogenous PMMoV using both mouse hepatitis virus (MHV) and Bovine Coronavirus (BCoV). They found that assay inhibition was present for RT PCR for all targets, endogenous or exogenous, even with the use of inhibitor removal columns and template dilution. Again, their results suggested that RT inhibition was increased relative to PCR in the RT PCR assay.  In the samples from plant A, but not from plant B, diluting the extract resulted in reduced inhibition.

Solids vs. Influent

The researchers, therefore, chose to use ddRT PCR for further analysis of wastewater solids. They found that when using different methods, the primary solids produced a higher number of SARS-CoV-2 signals with greater consistency compared to influent. The single-step ddRT PCR assay on the solids sample produced better results than the two-step assay, whether ddRT PCR or qRT PCR. This was their default method going forward.

They were able to detect N1 and N2 in 79 and 68 of the 96 samples, with mean values of 870 cp/g and 730 cp/g, respectively. Fecal strength was acceptably constant in the solids samples. They found that N1 and N2 concentrations correlated with the incidence of new cases in the area served by the wastewater plant involved. If the frequency of sampling and analysis is reduced to twice a week, significant associations are still obtained, but not if they are done only once a week or once a fortnight.

Choosing a Method

The researchers conclude that “Testing wastewater solids for SARS-CoV-2 may be more sensitive than testing influent.” The lack of detection in plant A samples could be due to the low COVID-19 prevalence in the area served. They also note that both N1 and N2 were detected together in almost all plant B settled solids samples but hardly ever did they co-occur in influent samples.

Again, they infer that the number of N1 and N2 copies in PCR-detected settled solids is much higher compared to influent since the concentrations are 350 to 3100 times higher in solids. The significance of this is that the use of solids in detecting the presence of SARS-CoV-2 in wastewater allows more sensitive detection at the community level, especially when the prevalence is low.

This method could be equally or more useful when it comes to viruses that have a greater affinity for wastewater solids, like adenovirus and rotavirus, since it can be used for epidemiological analysis of these viral illnesses as well. Another advantage is that it avoids the need for the preconcentration step.

When samples were collected over a more extended period, they found that the presence of N1 and N2 in solids was related to the number of new COVID-19 infections. They suggest that more plants will have to be evaluated to allow a more robust estimate to be produced.

However, they say, “The positive associations suggest that SARS-CoV-2 RNA in solids can be used to confirm trends in infection prevalence.” However, it is necessary to study more data on fecal shedding of the virus in order to understand if presymptomatic illness also links to viral RNA concentrations in solids.

The study also points out the need to test for inhibitors like ferric chloride in wastewater samples and to evolve methods to nullify such inhibition without the need for dilution.

Limitations and Future Directions

In the current study, the use of surrogate viruses promotes significant uncertainty since their agreement with SARS-CoV-2 in behavior, interaction with wastewater matrices, and similarity to the state of the endogenous viral target is all unknown.

However, they say, “We believe that recovery of a surrogate should be used to ensure that sample processing is not “out of the ordinary,” and can be used to identify problems during sample processing.”

Again, the methods used to reduce inhibition in the second longitudinal phase of the study also reduced the degree of virus recovery, explaining why the N1 and N2 concentrations were four-fold higher during the evaluation phase compared to the longitudinal phase.

The use of solids mandates the presence of a preliminary settling step in the sewage treatment plant, and this method cannot be applied in its absence, or when samples are obtained from manholes.

Future research may focus on how virus decay occurs within wastewater, both in influent and in solids, in order to uncover how viral RNA concentrations in these wastewater indices relates to the presence of infection in the area served by the plant. Again, the degree of mixing of different samples needs to be studied further to understand how far any given sample represents a true composite.

More data will also be required regarding how much and for how long viral RNA shedding occurs in feces, and the kind of viral particles or subparticles that are found, whether encapsulated virions or free RNA. This will help to understand how the virus survives and move in the sewer stream and thus to identify suitable detection methods.

*Important Notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Dr. Liji Thomas

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.


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