Assessment of a Program for SARS-CoV-2 Screening and Environmental Monitoring in an Urban Public School District

Key Points Question Does weekly testing of kindergarten through 12th grade students and staff improve detection of SARS-CoV-2 infection and understanding of the epidemiology of SARS-CoV-2 in urban public school settings? Findings In this quality improvement study, weekly school-based saliva polymerase chain reaction testing at 3 urban public schools was associated with increased case detection among staff and students compared with symptom-based strategies, exceeding county-level case rates. SARS-CoV-2 was detected in school wastewater samples each week as well as air and surface samples from choir classrooms. Meaning This study suggests that routine SARS-CoV-2 testing may identify infected staff and students who are not identified through conventional case detection and may provide insight into disease burdens of undertested communities.


eMethods.
Saliva extraction-free SARS-CoV-2 RT-PCR The Nebraska Extraction-free Saliva (NEfS) assay used for this program was optimized for high-throughput testing with considerations for minimizing overall testing costs, overcoming potential shortages of reagents, and decreasing turnaround time. In brief, 50 µL of well-mixed saliva is added to 6.3 µL of proteinase K (New England Biolabs, P8107S), then shaken and heated at 2,200 RPM and 95 o C for 5 minutes. Five microliters of the sample preparation are added to 15 µL of PCR master mix (TaqPath 1-Step RT-pPCR Master Mix, ThermoFisher Scientific A15299) containing primers and probes for detection of SARS-CoV-2 nucleoprotein RNA and human RNaseP RNA (Integrated DNA Technologies, IDT). RT-PCR is performed on QuantStudio7 Pro thermocyclers (ThermoFisher Scientific).
Wastewater processing and SARS-CoV-2 RT-PCR For RT-PCR testing, samples were processed using the Qiagen RNeasy PowerSoil Total RNA kit (Qiagen, 12866-25) following the manufacturer's instructions. For each sample, 70 mL of well-mixed wastewater was divided into two 50 mL conical tubes and centrifuged at 3,500 x g for 20 minutes to concentrate solids within the specimen. The supernatant was carefully removed, and the remaining pellet was resuspended in up to 2 mL of water and used as input for the PowerSoil extraction. VetMAX™ Xeno™ Internal Positive Control RNA (ThermoFisher Scientific, A29761) was spiked into each sample to verify extraction efficiency. SARS-CoV-2 was detected using the IDT 2019-nCoV RUO kit (IDT, 10006713). A standard curve was generated using a dilution series of quantitative synthetic SARS-CoV-2 RNA (ATCC, VR-3276SD). For total and suspended solid analysis, wastewater samples were processed following ASTM D5907-18.
Air and surface sample collection and processing for SARS-CoV-2 RT-PCR For air samples, cartridges were removed from the sampler and placed in a sealed bag for transport. Surface samples were collected using a prewetted (3 mL of sterile PBS) cotton swipe by wiping in an S-pattern, in two directions, over an approximately 100 cm 2 area. Following collection, surface swipes were placed into a 50 mL conical tub for transport. All air and surface samples were transported on ice to UNMC for processing. For air samples, the two metallic probes were removed from each cartridge and placed in a 15 mL conical tube with 10 mL PBS and then shaken by hand for one minute to liberate collected particles. Surface samples were recovered by adding 10 mL of PBS to the 50 mL conical tube and hand shaking for one minute. Following recovery, 400 µL of each recovered sample was extracted using the EZ1 Advanced XL Extractor (Qiagen, Hilden, Germany) with the EZ1® Virus Mini Kit v2.0. SARS-CoV-2 whole-genome sequencing RNA was extracted from saliva samples on a KingFisher Flex (ThermoFisher Scientific) using the MagMax Viral/Pathogen II (MVP II) Nucleic Acid Isolation kit (ThermoFisher Scientific, A48383) following the manufacturer's instructions for processing saliva samples. Samples were processed for sequencing by two different methods. For Oxford Nanopore Technologies (ONT) sequencing, the ARTIC multiplex PCR method V3 primer set was used following the publicly available protocol for amplification, library preparation, and analysis. 14 For Illumina-based sequencing, we used Swift Biosciences Normalase Amplicon SARS-CoV-2 V1 Panel 15 following the manufacturer's instructions. Libraries were sequenced on a 2 x 151 bp iSeq100 run. Genomes were assembled using the TAYLOR pipeline and consensus genomes were submitted to GISAID (EPI_ISL_1016947 -EPI_ISL_1016967).
The genomes from our study were aligned with 177 other genomes from Nebraska state, available on the GISAID repository (contributor acknowledgments provided in eTable 3) with MAFFT v1.4.0, as implemented in Geneious Prime v2021.0.3. A phylogenetic tree was inferred using RAxML v4.0 under the GTR+GAMMA model.

Statistical analyses
Data handling and analyses were performed in Microsoft Excel ® and SAS ® . Additional graphics were generated in GraphPad Prism 9 ® . In addition to descriptive epidemiology, each metadata element was assessed in univariate, and when appropriate, bivariate analyses for assessing impact on participants' first positive result. When statistically significant non-identity estimates of effect were identified or for presumed confounders that could be reasonably characterized, those elements were advanced to multivariate regression analysis. Community case rate data was not sufficiently granular to allow transforming zipcodes to levels of background COVID-19 risk. Socioeconomic status eTable 1.

Acknowledgment of Contributors to GISAID SARS-CoV-2 Genome Sequences Used in This Study
We gratefully acknowledge the following Authors from the Originating laboratories responsible for obtaining the specimens, as well as the Submitting laboratories where the genome data were generated and shared via GISAID, on which this research is based. All Submitters of data may be contacted directly via www.gisaid.org Authors are sorted alphabetically.

Genome entries
Originating laboratory Choir Number of cases identified (and so persons representing transmission risk and removed from in school activities) through the pilot in contrast to those identified by usual passive case capture.

Positive Cases Among Choir Students
A B C