Development and Validation of a Simplified Method for Analysis of SARS-CoV-2 RNA in University Dormitories

Abstract

Over the course of the COVID-19 pandemic, wastewater surveillance has become a useful tool for describing SARS-CoV-2 prevalence in populations of varying size, from individual facilities (e.g., university residence halls, nursing homes, prisons) to entire municipalities. Wastewater analysis for SARS-CoV-2 RNA requires specialized equipment, expensive consumables, and expert staff, limiting its feasibility and scalability. Further, the extremely labile nature of viral RNA complicates sample transportation, especially in regions with limited access to reliable cold chains. Here, we present a new method for wastewater analysis, termed exclusion-based sample preparation (ESP), that substantially simplifies workflow (at least 70% decrease in time; 40% decrease in consumable usage compared with traditional techniques) by targeting the labor-intensive processing steps of RNA purification and concentration. To optimize and validate this method, we analyzed wastewater samples from residence halls at the University of Kentucky, of which 34% (44/129) contained detectible SARS-CoV-2 RNA. Although concurrent clinical testing was not comprehensive, student infections were identified in the 7 days following a positive wastewater detection in 68% of samples. This pilot study among university residence halls validated the performance and utility of the ESP method, laying the foundation for future studies in regions of the world where wastewater testing is not currently feasible.

Figure 1:

A comparison of ESP with existing wastewater analysis processes^,,. Additional details on the comparator methods are given in a review by Rusiñol et al..

Figure 2:

Our residence hall sampling strategy involved two testing passes per week. In the first pass, clusters of buildings were sampled at downstream access points. A second testing pass refined the location of hotspots by sampling at access points representing single buildings.

Figure 3:

A) Photo of ESP device. B) Illustration of ESP operation, highlighting the movement of beads through a series of wells using magnets above and below the ESP well plate. C) Description of buffer and volume loaded into each of the labeled wells in Panel B.

Figure 4:

A) SeraSil captured significantly more SARS-CoV-2 RNA than other tested PMPs. *Stastistically significant as determined by 2-tailed Student’s t-test with Bonferroni correction. MagneSil and SeraSil-Mag both have lowest coefficients of variation of 38%. Error bars represent standard deviation. B) Capture efficiency of heat-inactivated SARS-CoV-2 (NR-52286) spiked wastewater. Error bars represent standard deviation.

Figure 5:

The relationship between wastewater SARS-CoV-2 RNA concentration (left-sided y-axis, blue line) and PCR-confirmed student SARS-CoV-2 infections (right-sided y-axis, orange line) during spring semester 2021 at residence hall clusters A-C. Note that the scales of the y-axes differ between graphs A-C. D) Correlation between wastewater measurements and the subsequent week’s clinical testing.