SARS-CoV-2 monitoring in sampling locations
SARS-CoV-2 monitoring through wastewater has been very informative and correlative to the number of COVID-19 cases. The SARS-CoV-2 wastewater surveillance in Washoe County, NV, was initiated early in the pandemic when the number of COVID-19 cases was starting to increase. Consequently, rising levels of SARS-CoV2 (detected through N1 and N2 genes) were observed in all three facilities influent wastewater since late September 2020, which correlated with an increase in the number of clinical COVID-19 cases in Washoe County 4,19. A detailed method of SARS-CoV-2 recovery from the wastewater was validated using current virus concentration methods by using the recovery control, inhibition control, and endogenous wastewater control 20. More than 600 samples from three facilities were analyzed over 15 months, and concentrations of gene copies ranged between 3.00 × 102 to 9.28 × 105 gc/L. We detected an increase in the concentrations of SARS-CoV-2 in wastewater with Ct values as low as 30.31, corresponding to 1.6x105 genome copied/L (Fig. 2 and Table S2). Therefore, we used wastewater samples from November 2020 onwards to detect SARS-CoV-2 signatures with an interval of 2 months (November 2020, January 2021, March 2021, and June 2021). SARS-CoV-2 variant signatures detected in wastewater were compared with the variants detected in COVID-19 clinical specimens during the same period.
Detection of SARS-CoV-2 variants through wastewater based epidemiology
While the viral load in the wastewater positively correlates with the number of SARS-CoV-2 infected individuals, determining the lineage of circulating variants can further provide intelligence on whether any specific variants change over time in the communities. SARS-CoV-2 variants impacting approved or authorized medical countermeasures or associated with more severe disease or increased transmission are categorized as variants of interests. However, when such variants are no longer detected or circulate at very low levels, posing minimal risk to public health, they are categorized under the monitored variants (VBM). Here, we hypothesized that SARS-CoV-2 variant signatures in wastewater positively correlate with circulating variants in humans. We tested our hypothesis by analyzing SARS-CoV-2 sequences in the clinical samples collected during the same period as the wastewater. Our site for the correlative study of variants in the wastewater and humans in the Reno-Sparks metropolitan area provides a decently controlled environment with a minimal influx of wastewater contribution from visitors, which may interfere with the signatures at a given time.
We began analyzing the wastewater sample of the most recent collection (June 20, 2021, for the presence of SARS-CoV-2 signatures, which identified many variants defining signatures of SARS-CoV-2 in the wastewater (Table S3). To determine the circulating variants, we generated a snapshot of SARS-CoV-2 variants present among individuals with SARS-CoV-2 infection by sequencing the clinical samples (N or NP swabs) of the Reno-Sparks metropolitan area at the NSPHL. SARS-CoV-2 sequences of the randomly selected clinical samples from the individuals of the Reno-Sparks metropolitan area during November 1, 2020, and June 30, 2021, were displayed through www.auspice.us (genomic epidemiology of novel coronavirus built on www.nextstrain/ncov) (Fig. 3). Lineages of SARS-CoV-2 circulated during the indicated months are highlighted on the phylogenetic tree (Fig. 3). The list of SARS-CoV-2 variants circulated during those months of wastewater genome surveillance are shown below the indicated months (Fig. 3). The diversity of variants decreased over time after introducing highly transmissible variants, B.1.1.7 and B.1.617.2. as expected due to the high transmission rate. Analysis of SARS-CoV-2 variants circulating in June 2021 was predominantly B.1.617.2 (Delta variant) with B.1.1.7 as the second predominant variant in the Reno-Sparks metropolitan area (Fig. 3). During March 2021, the most circulated variant was detected to be B.1.2, however lack of granularity for lower order lineage classification may have categorized them under lineage B.1.2. The other dominant variants, B.1.429 and B.1.1.7 during that time periods were classified under the category of VOIs or VOCs, respectively and these are currently under VBM. SARS-CoV-2 variants during January, 2021 and November, 2020, belonged to a large groups of variants (Fig. 3).
With an aim to maximize the detection of variants circulating in Washoe county, we performed enrichment and sequencing of SARS-CoV-2 of the wastewater during the above time period and compared with the variants detected through individual level testing of clinical specimens from the community. Lineage classification of variants in clinical specimens collected during June, 2021 showed predominantly B.1.617.2 (21A) and B.1.1.7 (20I), presented as a K-mer phylogenetic tree with relative proportions of each detected variants (Fig. 4). The variant signatures detected in wastewater through the variants calling tool of the CLC Genomic Workbench by comparing with Wuhan-Hu-1 SARS-CoV-2 are displayed in the associated_variant_track panel (Figure S2A). A list of all detected variants along with the count and coverage of each allele is presented (Table S3). The number of reads (count) and genome coverages for each samplewere determined using the CLC Workbench. Allelic frequencies (%) depicting the relative abundances, calculated based on the count and coverages of the reads, detected variants defining signatures of B.1.617.2 (21A) and B.1.1.7 (20I) clades (Fig. 4B). Relative prevalences of variants signatures in the wastewater showed a higher level of B.1.617.2 (21A) defining signatures, which is congruent with the percentages of SARS-CoV-2 cases determined by the individual level testing of clinical samples (Fig. 4C). Importantly, we were able to detect the signatures of low abundant variant, P.1 through the Wastewater Based Epidemiology (Fig. 4). This conclusively showed that WBE can detect the signatures of even low abundantly present SARS-CoV-2 variants and thus can be useful for detecting VOIs/VOCs signatures early in their spread.
Next, we determined whether the WBE functions is predictive of identifying variants over a period of time, by analyzing the diversity of SARS-CoV-2 variants in the wastewater collected at three additional time points, March, 2021, January, 2021 and November, 2020. The reads mappings and variant signatures detected in the wastewater collected during these months are shown as supporting data (Figures. S2B-D) along with the number of reads for each allelic variant including the variant-defining alleles (Tables S4-6). Relative abundances of SARS-CoV-2 variants detected through the individual level testing are presented as pie charts for those three indicated months (Figures. 5A, C and E). Allelic frequencies of variant-defining signatures in WBE were plotted as heat-maps, which showed the signatures of all the variants of interests as well as the variants of concerns (now variants being monitored) at three indicated time points (Figures. 5B, D and F). These variants are marked with red asterisks and the signatures of those VBM in the wastewater are marked above those mutations (Fig. 5). B.1.1.7 (20I) were among the predominant variants circulating during March, 2021 in Washoe County, which corelated with the highest number of reads for mutations defining B.1.1.7 (20I) variant (Figures. 5A and B). Similarly, B.1.427/429 (Epsilon) variants was highly prevalent in the month of January, 2021, also detected by the number of reads for the allelic mutations defining B.1.427/429 (Fig. 5C and D). We also detected the signatures of in B.1.1.7 through WBE in November, 2020, when the diversity of circulating mutations was high (Figures. 5E and 5F). This supports our hypothesis that WBE detects circulating SARS-CoV-2 variants in the community.
Comparison with SARS-CoV-2 variants monitoring in wastewater and the variants from clinical sequencing results
The predominant variants in the Washoe county during the month of June, 2021 were B.1.617.2, B.1.1.7 with a small proportion of B.1.526 and P.1 (Fig. 4). B.1.1.7, P.1 and B.1.617.2 belonged to the VOCs and B.1.526 to the VOIs, which are now under the category of VBM because of their low or almost no transmission but still requiring monitoring for their potential to countermeasure the approved therapeutics. When analyzing the frequencies of VBM signatures in the sequences of wastewater specimens, we found that the prevalences of these VOC/Is were correlated to their occurrences among community individuals. Percent frequencies of the reads with variants defining signatures for Alpha (B.1.1.7) and Delta (B.1.617.2) ranged between 80–90%, which was in the same range as the community prevalence of these variants during June, 2021 (Fig. 4). Similarly, during March 2021, Alpha (B.1.1.7) and Epsilon (B.1.429/427) were the most prevalent variants in the community, which was also reflected by the percent frequencies of these VBM specific signatures (Table S4). Lineage analysis of variants in the community during January 2021 showed a high diversity of variants with three VOIs (B.1.429/427 and B.1.526), which was also detected in wastewater samples collected during the same period (Table S5). Notably, the wastewater samples collected during November 2020, when only a few cases of the Alpha (B.1.1.7) variants in the Washoe County, were detected to contain mutations associated with the Alpha variant (Table S6). Although these signatures were not Alpha variant-specific and the other Alpha variant specific mutations (Spike, N501Y) were in the region with low sequencing coverage. We suspect that low coverage was due to the quality of RNA stored for over six months, as we retrospectively analyzed samples from earlier time points for this comparative study.