In this study, raw wastewater samples were collected from several sources. Among these sources, four were dormitories (Dorm A-D), one was an athletic facility (AF), one was an academic building (AB), and one was taken from a closed loop cooling water blowdown station that did not have any possible human contact as a negative control (CT) site. The source of wastewater collected for Dorm A was a small manhole pump station located immediately adjacent to the sewer outlet of the building, while the sources of the other dormitories, along with the athletic facility and academic building, were small lift stations. The manhole and lift stations temporarily stored raw wastewater discharged from each of the isolated buildings.

The investigation started on October 13, 2020, and continued until September 14, 2021. During this time, each sample location was collected 41 times, generally on a weekly basis. The sample timing was collected on a Tuesday or Wednesday morning selected to maximize the capture of fresh contributions from dormitory residents, athletes, and students. Weekends and afternoons were avoided to limit any possible impact of 1) visitors who might create false positives or 2) wastewater pumping that purged the station to create false negatives. Table 1 summarizes the sampling events conducted during the study period.

A total of 410 samples were collected during the study period. Sampling was conducted downstream first in order to avoid cross-contamination. Table 2 summarizes the documented occupancy in each of the dormitories for each time period of the study. The number of residents in each dormitory facility varied by semester from none to up to 1171.

All samples were grabbed in 250 mL sterile bottles ( Figure 2 ). A 15-ft long sampling pole was used to collect the samples from the wastewater in the wet well of each station. The samples were transferred to a 250 mL presterilized sample bottle. The pole was washed with deionized water immediately after sampling in a location to avoid cross-contamination. All collected samples were stored at 4ºC during the sampling events prior to analysis to minimize sample degradation. After collection, all wastewater specimens were brought to the laboratory for processing within 12 hours of collection.

IDEXX magnetic beads-based nucleic acid extraction kits were used for the RNA recovery, and an IDEXX water SARS-CoV-2 RT-PCR kit was employed for the molecular analysis using RT-qPCR. The sample concentration step was conducted following the manufacturer’s protocol. Briefly, a total of 105 mL of each wastewater specimen was subdivided into three centrifuge tubes (35 mL in each tube), which were then centrifuged at 4700G for 30 minutes at 4˚C. This step served to separate suspended solids from the liquid samples. Then, the supernatants were dissolved in 3.5 ± 0.1 g of polyethylene glycol (PEG) 8000 and 0.788 ± 0.01 g of sodium chloride (NaCl), as per Colombet et al. [64] . The prepared solutions were centrifuged at 12,000G for 2 hours at 4˚C followed by an additional centrifugation for 5 minutes at the same speed and temperature. The supernatant was discarded, and the pellets were resuspended in 400 µL of nuclease-free water. Therefore, 400 µL of concentrated samples were prepared for each of the representative wastewater specimens and stored at −20˚C until RNA extraction.

For the RNA extraction, IDEXX Magnetic Beads RNA extraction kits were used (Cat. No.: WCOV2MAG). The nucleic acid extraction was carried out according to the manufacturer’s protocol. At first, a working solution was prepared by mixing 500 µL of binding buffer (BB), 50 µL of proteinase K (PK), and 20 µL of magnetic beads (MB) in a micro-centrifuge tube for each sample. Then, 200 µL of the concentrated wastewater specimen was added to the working solution. The final solution was incubated at 58˚C for 10 minutes. Then, the magnetic beads were washed with 500 µL wash buffer-1 once and 500 µL of wash buffer-2 twice for each sample to remove inhibitors, proteins, and other contaminants. A magnetic rack was used to discard the liquid solution while keeping the beads in the micro-centrifuge tube. Finally, 100 µL of elution buffer was used to dissolve the purified viral RNA after incubating the washed magnetic beads at room temperature for 10 minutes. As a result, 100 µL of viral RNA solution was prepared for each raw wastewater sample. The extracted viral RNA was stored at −20˚C until the PCR assay.

IDEXX RT-PCR kits (Cat. No.: WCOV2PCR) targeting the N3 gene of the SARS-CoV-2 were employed for the molecular analysis. The PCR reporter used in this kit is FAM, while ROX is employed as the passive reference. The quencher used in the kit is BHQ^®. The RT-PCR step was conducted following the manufacturer’s instructions. At first, 10 µL each of the SARS-CoV-2 mix and RNA master mix were added, followed by the addition of 5 µL of representative recovered RNA to make the final volume of each reaction 25 µL. For the positive control (PC) and negative target control (NTC), 5 µL each of PC and PCR grade water were added respectively in place of the template. The experiments for each of the samples were carried out in duplicate. Finally, the product was amplified following the thermal cycle described in Table 3 .

After PCR amplification, C_q values were recorded for each reaction. The final C_q value for a specific wastewater RNA solution was measured by taking the average C_q values obtained from the duplicate reactions. To quantify the number of RNA copies as well as for quality assurance purposes, a four-point standard curve was prepared after obtaining the C_q values of serially diluted standard samples with known numbers of gene copies. The coefficient of determination (R²) for the prepared curve was 0.9987, indicating a strong correlation. The equation obtained from the standard curve was used to evaluate the log copies for each of the tested RNA solutions. From the prepared curve, the detection limit for the approach was found to be 40 RNA copies/100 mL of sample. Finally, the number of viral RNA copies for each of the reactions was calculated using the following mathematical expression:

$\begin{array}{l}\frac{Viralcopies}{100mL}={\log }^{-1}\left(\log copiesperreaction\right)\\ \times \frac{ExtractedRNA\left(\mu L\right)}{PCRRNA\left(\mu L\right)}\times \frac{\mathrm{Pr}eparedconcentrate\left(\mu L\right)}{Usedconcentrate\left(\mu L\right)}\times \frac{100}{Initialsample\left(mL\right)}\end{array}$

Where the extracted RNA is the volume of recovered RNA (100 µL), PCR RNA refers to the volume of RNA solution used in each reaction (5 µL), prepared concentration is the total volume of concentrated samples for each wastewater specimen (400 µL), used concentrate is concentrated used for the RNA extraction (200 µL), and finally, initial sample refers to the wastewater specimens used for the concentration step (105 mL).

In the current study, two types of clinical data were used. The number of infected individuals occupying Dorm A, Dorm B, Dorm C, and Dorm D were regularly monitored by university administration officials. Clinical diagnostic tests were conducted within the campus for symptomatic patients as well as for new students and student residents returning to campus from breaks. This data was tracked by the university. The infected number of residents/guests from the dormitories each day was taken into consideration for further analysis. Since the number of infected individuals was not routinely or systematically monitored for the AF and AB facilities during the time of the study, the WBE outcomes for the sampling sites were also compared with the 7-day rolling averages of the broader infected population in Palm Beach County. The 7-day rolling averages of the number of infected cases in Palm Beach County were retrieved from USA Facts [65] .

The time series data of the obtained WBE outcomes, along with the number of COVID-19 cases identified from the four isolated dormitories recorded from October 13, 2020, to September 14, 2021, are summarized in Figure 3 . During fall 2020, the pandemic lockdown was lifted at the university, but classes were still being conducted in online format only. However, dormitories were reopened for students who chose to reside on campus. In the case of Dorm A, a sharp spike in viral signal was observed on October 20, 2020, with 275 viral RNA copies/100 mL. In the following week of this peak result (October 27, 2020), the 7-day rolling average of infected individuals was greater than one (1.1). As expected, immediately after returning from the winter break to start the spring 2021 term, spikes were observed for all dormitories. Classes were still only being offered in fully online format throughout the spring 2021 term. The maximum viral RNA concentration was seen on January 29, 2021 (291 copies/100 mL), while a 25% increase in the 7-day rolling average was observed (0.57 in January 27, 2021 to 0.71 in January 29, 2021). A similar phenomenon was also observed for Dorm B (0.29 to 0.85) and Dorm D (0.29 to 0.71) ( Figure 3b and Figure 3d ). This can be attributed to students and their parents/friends arriving, onboarding, moving in, and transitioning later than usual. In addition, the university was closed on January 18, 2021, in observance of the Martin Luther King Jr. federal holiday. During spring 2021, semester maximum viral concentrations were found on March 16, 2021, for Dorms B, C, and D with 10,680, 34,520, and 4,130 copies/100 mL, respectively. The 7-day rolling averages of COVID-19 cases were also found to be about 50% higher in the following week (March 23, 2021) compared to the previous one. This is attributed to the residents returning from mid-semester break (March 5 - 15, 2021) in which the opportunities for infection were greater due to vacation travel exposures. Later, vaccines became widely available for the residents from mid-April 2021 (being limited to age- and occupation-related restrictions prior to this date), and both the numbers of infected individuals and viral RNA concentrations were found to decrease thereafter.

During the break between the spring and summer terms, no spikes were observed in Dorms A, B, and D. In Dorm C, only one individual tested SARS-CoV-2 positive, while the number of viral RNA copies was 103 copies/100 mL for the wastewater sample collected on May 4, 2021 ( Figure 3c ). In the summer 2021 term, fewer numbers of residents remained in the dorms, and as expected many of the samples were below detection. The maximum viral RNA

concentrations were determined as 220, 7340, 360, and 477 copies/100 mL for Dorm A, Dorm B, Dorm C, and Dorm D, respectively, during the summer 2021 term. Most of the positive WBE results were obtained during the second half of the summer term. This coincided with the new summer admits and freshman class arriving on campus in anticipation of the campus fully reopening to in-person coursework. The 7-day rolling averages were below 0.2 cases during most of the term. In the meantime, the Delta Variant wave began to dominate in South Florida from the beginning of July 2021, and upward trends were observed for the number of infected students from the second week of the month. During fall 2021, coursework began to transition back to in-person (face-to-face) modalities. Although no correlation was observed between the number of viral RNA copies and 7-day rolling averages of infected individuals, the trends between viral copies and infection incidence remained similar, just as reported in studies conducted at the University of Arizona [62] and the University of Miami [66] using RT-PCR testing.

The current study also tested samples collected from the lift stations of the school’s main athletic facility (AF) as well as from one isolated academic building (AB). Figure 4 summarizes the outcomes of the WBE results for the two sampling locations along with the 7-day rolling average of the infected population in Palm Beach County. An athletic event took place on October 31, 2020, and the measured viral concentration for the following week (November 4, 2020) was found to be 52 copies/100 mL. In most of the WBE results in fall 2020, the athletic facility sample was observed as SARS-CoV-2 positive, as the athletic teams had a busy training and game schedule during that time.

The graduation ceremony for the spring 2021 term took place on April 29, 2021, outdoors in the athletic facility, where almost 3200 degrees were awarded in two in-person commencement ceremonies. In addition, graduates were provided with up to 5 tickets for family members to attend, and several hundred volunteers, security personnel, paramedics, faculty, and staff were also in attendance. As expected, there was a discernible uptick on May 4, 2021, with an elevated RNA concentration of 80 copies/100 mL compared to the several weeks prior when the facility was not being utilized as heavily. Spikes were also observed at the AB for the fall 2020, spring 2021, and fall 2021, especially during the beginning of the terms. The rise in the WBE results for the AB may have happened due to the fact that faculty members and students used offices and lab facilities of the building throughout the semesters, although no in-person classes were offered in fall 2020 and spring 2021. During the semester breaks, no spike was observed for the AB, likely because the occupancy of the building was low during the holidays.

The current study collected a total of 41 weekly wastewater samples from each of the four student dormitories from October 13, 2020, to September 14, 2021. The RT-PCR results were compared with the diagnostic test results. A summary of the comparison between the clinical data and positive/negative outcomes is presented in Table 4 and Table 5 , respectively. From Table 4 , the samples collected from Dorm C demonstrated maximum positive results (68.3%), while Dorm A had the minimum (43.9%). In most cases, the number of infections for a specific dormitory followed the WBE outcomes for the previous week. In the case of Dorm C, the WBE approach provided correct prediction nearly 80% of the time for the positive and negative cases, one week before the diagnostic test results. There were only a few sporadic incidents where positive cases occurred in the following weeks after a non-detect WBE result for the studied dormitories – 12, 11, 5, and 8 times for Dorm A, Dorm B, Dorm C and Dorm D, respectively. These incidents decreased when the data was compared with the infected cases confirmed in the two days following sample collection from each dormitory (4, 6, 2, and 3 times for Dorm A, Dorm B, Dorm C, and Dorm D, respectively). The samples for Dorm A were collected from a manhole, where the duration of wastewater storage was relatively lower when compared to the residence time in the other lift stations, which during the shutdown, could have been days for the AB since few people were using the facilities.

In contrast, in 52% of the instances where the WBE result was below detection, there was at least one infection in Dorm A. In the case of Dorm B, 11 out of the 17 below detection WBE results were followed with at least one positive case the next week, and 6 of them occurred in the following few days. However, in nearly all (5 of these 6) scenarios, the number of infected individuals was only one. It is reasonable to suppose that a portion of infected individuals might not shed the virus, or could have lower virus shedding, or might have shed the virus while in another location, or the viral copies might have been transported downstream before sampling [62] . In all three other occurrences where positive clinical cases were observed within 2 days after collection, the number of infected individuals was one. On the other hand, below detection results for Dorm C and D provided accurate predictions in nearly all cases.

Overall, the positive predictive value of the WBE approach conducted in the current study was 86% considering the number infected individuals in the following one week after sample collection. In Betancourt et al. [62] , sensitivity and specificity were 76% and 90.7%, respectively, considering the diagnostic test results obtained within 4 days of the wastewater sample collection. The approach enabled early detection of 85% of COVID-19 cases in the study of Karthikeyan et al. (2021) carried out at the University of California, while the study of Betancourt et al. [62] successfully predicted 82% and 88% of the positive and negative cases respectively after four days of the sampling. In the case of the control sample used in this study, none of the collected specimens showed positive results, as expected.

The Spearman correlation coefficient was used to evaluate the relationship strength of the number of RNA copies/100 mL results from the WBE analysis and the collected data of the 7-day rolling average of the infected individuals for each of the dormitories. The current study also observed the Spearman correlation coefficient after comparing the data with the documented COVID-19 cases on the next day of sampling. The relationships are summarized in Table 6 , which describes a moderate correlation for Dorm B, C, and D and a weak relationship for Dorm A (which was located furthest upstream and had the smallest wastewater storage volume capacity of any of the sampling locations). For comparison, a study conducted at the University of Miami [65] found that the value of r_s was 0.39, while comparing the results obtained by qPCR (WBE approach) with the 7-day rolling average for the same week.

Positive r_s values for each dormitory location suggest that an increase in the RNA copies was generally followed by a subsequent rise of the 7-day rolling average for the following week. The other three dorms tested showed relatively stronger correlations between the WBE outcomes and COVID-19 cases for the following week. The statistical analysis of the WBE outputs and clinical cases of the next days of the sampling events showed a stronger relationship with r_s values of 0.18, 0.51, 0.51, and 0.48 for Dorm A, Dorm B, Dorm C, and Dorm D, respectively. Table 6 also lists the P-values, which demonstrate the significance of

the relationships. The P values of 0.06 and 0.007 for the following day and the following week of the sampling events, respectively, represent high significance of the data obtained for Dorm A. The maximum P values were observed for Dorm C (0.07 for both the next day and the next week of the sampling), indicating the moderate significance of the outcomes.

The probability of obtaining a positive WBE result after comparing it to the number of infected individuals in the following 7-day period after the sampling is summarized in Table 7 . For Dorm A, there were 19 occurrences when the number of infected individuals was confirmed to be between 1 and 4 in the week following sample collection, with positive WBE results found eight times during these incidents.