SARS-CoV-2 Titers in Wastewater Are Higher than Expected from Clinically Confirmed Cases

Abstract

Wastewater surveillance represents a complementary approach to clinical surveillance to measure the presence and prevalence of emerging infectious diseases like the novel coronavirus SARS-CoV-2. This innovative data source can improve the precision of epidemiological modeling to understand the penetrance of SARS-CoV-2 in specific vulnerable communities. Here, we tested wastewater collected at a major urban treatment facility in Massachusetts and detected SARS-CoV-2 RNA from the N gene at significant titers (57 to 303 copies per ml of sewage) in the period from 18 to 25 March 2020 using RT-qPCR. We validated detection of SARS-CoV-2 by Sanger sequencing the PCR product from the S gene. Viral titers observed were significantly higher than expected based on clinically confirmed cases in Massachusetts as of 25 March. Our approach is scalable and may be useful in modeling the SARS-CoV-2 pandemic and future outbreaks.IMPORTANCE Wastewater-based surveillance is a promising approach for proactive outbreak monitoring. SARS-CoV-2 is shed in stool early in the clinical course and infects a large asymptomatic population, making it an ideal target for wastewater-based monitoring. In this study, we develop a laboratory protocol to quantify viral titers in raw sewage via qPCR analysis and validate results with sequencing analysis. Our results suggest that the number of positive cases estimated from wastewater viral titers is orders of magnitude greater than the number of confirmed clinical cases and therefore may significantly impact efforts to understand the case fatality rate and progression of disease. These data may help inform decisions surrounding the advancement or scale-back of social distancing and quarantine efforts based on dynamic wastewater catchment-level estimations of prevalence.

Keywords: COVID-19 prevalence; PMMoV; SARS-CoV-2; viral titers; wastewater.

FIG 1

Sanger sequencing results and alignment to SARS-CoV-2 S gene. Sequence was aligned to SARS-CoV-2 S gene (GenBank MT577641.1) using NCBI BLASTN. Highlighted gray, forward and reverse primers; highlighted yellow, aligned sequence; highlighted purple, mismatched sequence from either primer. The first 24 bases in the COVID-19 S gene are the T7 promoter added by PCR with S-F and S-R primers in Table 1. Results for the Southern influent sample were similar. Sequencing chromatograms for forward and reverse primers are provided in Fig. S1B.

FIG 2

Estimated viral titer per milliliter of sewage. Estimated gene copies in sewage samples for each of the three CDC primer sets are shown for all dates. Northern (N) and Southern (S) influents are shown separately, except for biobanked January samples, which are combined.

FIG 3

Sample stability over time. Estimated copies per milliliter of sewage estimated from the same samples filtered and processed on different dates. The y axis shows samples processed between 20 and 25 March 2020, and these are plotted against samples reprocessed on 4 April 2020 (samples were pasteurized upon receipt and stored at 4°C until processing). The gray dashed line corresponds to y = x. Each point represents an average over all three primer sets for each sample.

FIG 4

PMMoV normalization for sewage samples collected from 18 March to 25 March. (A) Original SARS-CoV-2 titers averaged from N1, N2, and N3 primers in the sewage sample. (B) Relative SARS-CoV-2 titers against PMMoV concentration in each of the samples. Data shown are averaged from N1, N2, and N3 primers. Influent samples from Northern (N) and Southern (S) influents are shown separately for each date.