Monitoring Trends of SARS CoV-2 and the Emergence of its Variants in a College Community via Wastewater Based Epidemiology

INTRODUCTION Wastewater-based COVID-19 monitoring programs around the world are currently ongoing with universities supporting the surveillance of their communities including in some cases their college (6). Many of these programs were initiated in 2020 to address early warning of disease amongst college students with a goal of serving as a complementary tool for public health agencies, yet the communication with the local health units is not always undertaken. The monitoring of wastewater to address infections may be advantageous because it is noninvasive and appears to be a leading indicator that precedes confirmed clinical cases (1). Wastewater Based Epidemiology (WBE) is considered cost effective as presymptomatic and asymptomatic individuals, in addition to symptomatic individuals can be monitored which can facilitate real time quantitative surveillance of the presence and spread of COVID-19 in a community (4). Currently, with the introduction of vaccination strategies in the community, WBE may serve as a vital tool to cut down on extensive clinical testing while implementing broad wastewater-based surveillance capabilities. Emergence of variants of concern (VOCs) such as the Alpha and Delta suggests that sustained vigilance is required to control the COVID-19 pandemic (5). The potential public health challenges that the emerging VOCs pose warrant extensive real time monitoring at the community level to track the timing and local spread of variants to inform efforts in controlling the pandemic (2). The objectives of this study were to investigate the spread of COVID 19 between the community and a college population using wastewater surveillance of SARS CoV-2 during various stages of the pandemic; the potential of WBE to serve as a leading indicator of SARS CoV-2 infections, and to inform spatial and temporal trends of viral spread in the college community. The study highlights how WBE can serve as an early warning to track the influx of new variants of concern in a population and their prevalence and spread in a small community. METHODS Wastewater samples (500-1000 mL) for this study were collected from 19 sanitary sewer sites. The dormitories and residential areas (n=15) were considered primary focal points for monitoring on campus and four additional locations in the neighboring community of East Lansing. A total of 557 samples were collected, twice a week, using composite autosamplers (between 5am and 12pm) from January through September 2021. All samples were kept at 4°C and processed within 6 hours of collection. 100mL samples were mixed with 8% polyethylene glycol 8000 and 0.2 M NaCl. The samples were mixed slowly on magnetic stirrer at 4°C/2 hours and then held at 4°C/0-16 hours. Following the incubation step, samples were centrifuged at 4,700 x g for 45 mins at 4°C. The supernatant was then removed by aspiration, and the pellet resuspend in the remaining liquid. Viral ribonucleic acid (RNA) was extracted from wastewater concentrates using the QIAmp Viral RNA Minikit (Qiagen, Germany) according to the manufacturers protocol with modifications. The modification included extraction of a total of 200 μL of concentrate which resulted in a final elution volume of 80 μl. SARS-CoV-2 RNA was quantified using the one-step RT-ddPCR approach. Two SARS-CoV-2 markers were chosen for analysis in duplex, the nucleocapsid (N1) and (N2) gene targets (2). Droplet digital PCR was performed using Bio-Rad's 1-Step RT-ddPCR Advanced kit with a QX200 ddPCR system (Bio-Rad, CA, USA) according to procedures as per Flood et al; 2021 (2). Molecular detection/quantification of SARS-CoV-2 virus (S gene wild type and variant for N501Y, del69-70, T478K and L452R mutation targets) was carried out with the GT-ddPCR SARS-CoV-2 Variants of Concern Mutational Signature Assay Kit (GT Molecular, Colorado, USA) kit as per the kit instructions using 5ul of RNA template per reaction well and quantified as described above. RESULTS AND DISCUSSION The monitoring effort included a total of 557 samples, which were collected between January and September 2021 at 19 different sites serving the campus and its nearby communities. Fifteen dormitory sites were monitored, and 430 samples were analyzed for the N1 and N2 gene targets. Positivity rates of 36-64% were observed at the dormitory sites where the cumulative percent positives at all the sites was 49% (211/430). The average concentrations obtained at the 15 dorm sites ranged between 1.25x103 - 6.34x104 gene copies/100mL. Four sites representing off campus community wastewater yielded a 61% (77/127) positivity rate with the average concentrations ranging between 1.16x103 and 5.09x103 gene copies/100mL. The influx of variants of concern, Alpha and Delta was tracked using targeted mutation assays which detected the signature mutations, N501Y/del69-70 and T478K/L452R respectively. Samples were analyzed for the presence of the Alpha variant beginning January 2021. The Alpha variant was first quantified at the community site (MSU-3) on March 15th, 2021. Two days later, the variant was detected at the MSU-6 and MSU-10 dormitory locations indicating possible spread from the neighboring community. The variant was detected in 12% (61/531) of the samples tested between January and September 2021 at 11 of the 19 locations monitored. The positivity rates for the Alpha variant at these locations ranged between 8 and 28%. Monitoring for the Delta variant began in July 2021 and the first quantification of its signature mutations was at the community site MSU-13 on 14th July 2021. Subsequently, 2 weeks later samples from other community sites, MSU-3 and MSU-12 also showed the presence of the Delta variant mutations. The first quantifiable detection of the Delta variant on campus was seen at dorm MSU-6 on 11th August 2021; 27 days after it was detected in the East Lansing community. All 19 sites showed Delta variant detections in the following weeks at positivity rates between 12 and 59%. Thirty-five percent (82/234) of all the samples analyzed between July and September 2021 were positive for the Delta variant mutations. CONCLUSION The trends observed through SARS CoV-2 wastewater-based surveillance indicated that the viral prevalence tracked well with the clinical infections in the community. The influx of variants of concern, Alpha in early 2021 and then Delta in mid-2021 were monitored and trends of SARS CoV-2 lineage shifts from Wildtype to Alpha and then to the current dominant Delta were studied on the college campus and in the neighboring community. The arrival of students on campus after Spring break in March 2021 and at the beginning of the Fall semester in Aug 2021, saw increased incidences of viral detection in the wastewater, the widespread transmission of COVID-19 and the variants of concern Alpha and Delta. Hence, WBE has shown promise as a tool to understand the spread of SARS CoV-2 and its variants on a campus which first appeared in the community and served as a sentinel system of the risk.