Enumerating asymptomatic COVID-19 cases and estimating SARS-CoV-2 fecal shedding rates via wastewater-based epidemiology

Background: Multiple jurisdictions around the world have attempted to quantify the total number of individuals infected with SARS-CoV-2 in the community that excreted the virus into wastewater (Ahmed et al., 2020; Chavarria-Miro et al., 2021; Curtis et al., 2020). Models and predictions have been based on fecal shedding rates derived from limited results that indicated a large variance across a small number of individuals (Wolfel et al., 2020). Furthermore, these fecal shedding rates were extrapolated from the 90th percentile of this limited dataset and used to calculate the number of infected individuals contributing to viral loads in wastewater (Ahmed et al., 2020; Chavarria-Miro et al., 2021; Curtis et al., 2020). However, these estimates are likely to be inaccurate without pairing wastewater results with clinical data. More recently, the University of Arizona (UArizona) used WBE, dovetailed with targeted clinical testing, to prevent COVID-19 outbreaks in student dormitories (dorms) (Betancourt et al., 2021). The current study builds upon the successful public health actions performed at UArizona by aggregating data from positive wastewater samples with number of clinically positive cases to provide estimations for fecal shedding rates amongst infected university students. This also enabled tracking the proportion of symptomatic and asymptomatic individuals that corroborated with wastewater results from defined communities composed of young adults (18-20 years old). This is the first study to determine community-wide fecal shedding rates by directly pairing clinical and wastewater results. Findings from this study may act as a benchmark to assess SARS-CoV-2 prevalence in a population and inform public health prevention and response actions. Methods: In total, 13 student dorms (Dorm A-M) at UArizona main campus were monitored throughout the Fall 2020 Semester (August 17--November 20). Wastewater samples were collected two-times-per-week from sewer manholes specific to each dorm's effluent prior to convergence with other sewer pipelines; thus, all wastewater samples were specific to the defined communities living in each dorm. The total number of residents at each dorm was known (Table 1) and was considered a closed population as visitors were prohibited. Positive detection of SARS-CoV-2 RNA (via the N1 gene region) in wastewater acted as the leading indicator for the presence of SARS-CoV-2 infections within the dorm communities. Results were immediately communicated to the UA Task Force and Campus Re-Entry Working Groups, which planned and conducted clinical testing of residents (Betancourt et al., 2021). Clinical tests for COVID-19 diagnosis were performed via antigen testing from nasal swab samples and RT-PCR from nasopharyngeal swab samples. Individuals with COVID-19 symptoms who sought clinical testing were tested through Campus Health Services (CHS), while non-reporting and asymptomatic cases were tested via targeted clinical testing at dorms with positive wastewater detection through the Test All Test Smart Program (TATS). Individuals that tested positive were removed from the dorm and transferred to another location for isolation. To estimate the number of SARS-CoV-2 infected individuals who contributed to a single positive wastewater sample, a 6-day range of clinical data was considered. Positive clinical cases from the day before, day-of, and four days after sampling were included in the count of infected individuals contributing to viral shedding. The rationale for this approach is: 1) Residual virus-shed from individuals testing positive for COVID-19 the day prior to sampling-may be detected in wastewater the following day. Previous sampling indicates that virus can persist for extended periods of time and be detected even when an estimated 1000 gallons of wastewater has continued to flow through a sewer system (Betancourt et al., 2021). 2) Individuals infected with SARS-CoV-2 may shed virus into wastewater prior to showing symptoms and/or being identified as a clinical case. The viral load of SARS-CoV-2 appears to peak in the upper respiratory tract of infected individuals within the first week of infection (Cevik et al., 2020) and the median incubation period for COVID-19 is estimated to be approximately five days (Lauer et al., 2020). Although infected individuals can shed virus several weeks after infection (Cevik et al., 2020), the 6-day window for calculating shedding rates was appropriate since wastewater testing was performed at least twice per week and all infected individuals that tested positive within the 3-4 day window between samples were removed into isolation prior to the next sampling event. Also, individuals were assumed to be at peak shedding for the first 6 days in which an infected person could be identified via UArizona WBE protocols. The fecal shedding rate of SARS-CoV-2 RNA per gram of feces from an infected individual was enumerated based on known concentrations of viral RNA in positive wastewater samples and the fact that the total numbers of infected individuals contributing to the total virus load in the samples were also known. The fecal shedding rate (FS) defined as genome copies per gram-feces (gc/g-feces) was calculated using Equation 1 based on the N1 gene concentrations. Results and Discussion: This current study examined clinical testing data to determine the abundance of asymptomatic versus symptomatic cases in these defined communities. For all dates in the study period, a total of 711 clinical cases were reported among the 13 unique dorms. Across all dorms, 148 symptomatic cases and 563 asymptomatic cases were reported; thus, 79.2% of SARS-CoV-2 infections were asymptomatic and only 20.8% were symptomatic (Table 2). Positive wastewater samples were aligned with clinical data to extrapolate the fecal shedding rates of SARS-CoV-2 N1 gene. The average N1 shedding rate per infected person was calculated to be 7.30 ± 0.67 log10 gc/g-feces, considering all positive wastewater samples across all dorms (Table 3). Until this research, fecal shedding rate of SARS-CoV-2 from infected individuals had not been well understood. This study successfully enumerated fecal shedding rates in young university students (18-20 years old) which can be used in models for estimating disease prevalence. Our shedding rate figure is an assumed early infection shedding rate estimate because students were identified within the 6-day period and transferred from the dorm into an isolated living quarters. Sensitivity analyses performed in this study suggest that detected RNA concentrations in wastewater samples is the most influential factor for estimating viral shedding rates in infected individuals. Other factors such as demographics of a specific community (i.e., median age) and disease severity may also influence viral shedding rates in feces. Although the approximate shedding rate from this study does not apply for the general population, this project provides a blueprint for conducting further investigations to determine viral shedding rate variance among specific demographics. Our team is currently conducting such analysis on six communities across the USA with median age groups ranging from 27 to 62 years of age (Table 4). Preliminary data suggests that community-wide shedding rates increases with median age groups (Figure 2). Final results and interpretations from this investigation are anticipated by end of 2021 and will be ready to share at the conference. These findings will provide shedding rates based on population demographics, which are essential for building disease prevalence models for specific communities. Lastly, it is important to note that no fecal samples were collected from individual COVID-19 patients directly. Therefore, results in this study are specific to wastewater from defined communities. These data are valuable due to the limited clinical research and evaluations on SARS-CoV-2 fecal shedding rates. Concluding Remarks: This study may have significant implications for public health. The fecal shedding rate of SARS-CoV-2 RNA derived in this study may be incorporated into models to estimate the total number of infections in communities with similar demographics based on wastewater viral concentrations. Knowledge of disease prevalence, especially as a leading indicator, can be used to assist communities in efficient resource allocation to prevent and contain COVID-19 outbreaks. This study also provides further understanding for the total number of cases that are symptomatic versus asymptomatic. Moving forward, the number of reported cases can provide context for estimating the number of cases that were asymptomatic and/or unreported. This, in turn, could have implications for understanding the proportion of individuals that has been exposed to COVID-19 and for understanding progress towards immunity within a community.