Wastewater-Based SARS-CoV-2 Surveillance in Northern New England.

SARS-CoV-2 viral RNA is shed in the stool of 55-70% of infected individuals and can be detected in community wastewater up to 7 days before people present with COVID-19 symptoms. The detection of SARS-CoV-2 RNA in wastewater may serve as a lead indicator of increased community transmission. Here, we monitored viral concentrations in samples collected from nine municipal wastewater facilities in New Hampshire (NH) and Vermont (VT).Twenty-four-h composite primary influent wastewater samples were collected from nine municipal wastewater treatment facilities twice per week for 5 months (late September 2020 to early February 2021). Wastewater was centrifuged for 30 min at 4600 × g, then the supernatant was frozen until further analysis. Once thawed, samples were concentrated, extracted, and tested for SARS-CoV-2 RNA using reverse transcriptase-quantitative PCR (RT-qPCR) and reverse transcriptase-droplet digital PCR (RT-ddPCR) detection methods. Active case counts for each municipality were tracked from the NH and VT state COVID-19 dashboards. We received a total of 283 wastewater samples from all sites during the study period. Viral RNA was detected in 175/283 (61.8%) samples using RT-qPCR and in 195/283 (68.9%) samples using RT-ddPCR. All nine sites showed positivity in the wastewater, with 8/9 (88.8%) sites having over 50% of their samples test positive over the course of the study. Larger municipalities, such as Nashua, Concord, and Lebanon, NH, showed that SARS-CoV-2 positivity in the wastewater can precede spikes in active COVID-19 case counts by as much as 7 days. Smaller municipalities, such as Woodsville, NH and Hartford, VT, showed sporadic SARS-COV-2 detection and did not always precede a rise in active case counts. We detected SARS-CoV-2 RNA in samples from all 9 municipalities tested, including cities and small towns within this region, and showed wastewater positivity as an early indicator of active case count increases in some regions. Some of the smaller rural municipalities with low case counts may require more frequent sampling to detect SARS-CoV-2 in wastewater before a case surge. With timely collection and analysis of wastewater samples, a community could potentially respond to results by increasing public health initiatives, such as tightening mask mandates and banning large indoor gatherings, to mitigate community transmission of SARS-CoV-2. IMPORTANCE Despite vaccination efforts, the delta and omicron variants of SARS-CoV-2 have caused global surges of COVID-19. As the COVID-19 pandemic continues, it is important to find new ways of tracking early signs of SARS-CoV-2 outbreaks. The manuscript outlines how to collect wastewater from treatment facilities, concentrate the virus in a dilute wastewater sample, and detect it using two sensitive PCR-based methods. It also describes important trends in SARS-CoV-2 concentration in wastewater of a rural region of the United States from Fall 2020 - Winter 2021 and demonstrates the utility of wastewater monitoring as a leading indicator of active SARS-CoV-2 cases. Monitoring changes in concentration of SARS-CoV-2 virus in wastewater may offer an early indicator of increased case counts and enable appropriate public health actions to be taken.

CRISPR-cas13 enzymology rapidly detects SARS-CoV-2 fragments in a clinical setting.

BACKGROUND: The well-recognized genome editing ability of the CRISPR-Cas system has triggered significant advances in CRISPR diagnostics. This has prompted an interest in developing new biosensing applications for nucleic acid detection. Recently, such applications have been engineered for detection of SARS-CoV-2. Increased demand for testing and consumables of RT-PCR assays has led to the use of alternate testing options. Here we evaluate the accuracy and performance of a novel fluorescence-based assay that received EUA authorization for detecting SARS-CoV-2 in clinical samples. METHODS: The Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) technology forms the basis of the Sherlock CRISPR SARS-CoV-2 kit using the CRISPR-Cas13a system. Our experimental strategy included selection of COVID-19 patient samples from previously validated RT-PCR assays. Positive samples were selected based on a broad range of cycle thresholds. RESULTS: A total of 60 COVID-19 patient samples were correctly diagnosed with 100% detection accuracy (relative fluorescence ratios: N gene 95% CI 29.9-43.8, ORF1ab gene 95% CI 30.1-46.3). All controls, including RNase P, showed expected findings. Overall ratios were robustly distinct between positive and negative cases relative to the pre-established 5-fold change in fluorescence. CONCLUSIONS: We have evaluated the accuracy of detecting conserved targets of SARS-CoV-2 across a range of viral loads, including low titers, using SHERLOCK CRISPR collateral detection in a clinical setting. These findings demonstrate encouraging results, at a time when COVID-19 clinical diagnosis and screening protocols remain in demand; especially as new variants emerge and vaccine mandates evolve. This approach highlights new thinking in infectious disease identification and can be expanded to measure nucleic acids in other clinical isolates.

High-Throughput CRISPR-Cas13 SARS-CoV-2 Test.

BACKGROUND: The ability to control the spread of COVID-19 continues to be hampered by a lack of rapid, scalable, and easily deployable diagnostic solutions. METHODS: We developed a diagnostic method based on CRISPR (clustered regularly interspaced short palindromic repeats) that can deliver sensitive, specific, and high-throughput detection of Sudden Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2). The assay utilizes SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) for the qualitative detection of SARS-CoV-2 RNA and may be performed directly on a swab or saliva sample without nucleic acid extraction. The assay uses a 384-well format and provides results in <1 hour. RESULTS: Assay performance was evaluated with 105 (55 negative, 50 positive) remnant SARS-CoV-2 specimens previously tested using Food and Drug Administration emergency use authorized assays and retested with a modified version of the Centers for Disease Control and Prevention (CDC) quantitative PCR with reverse transcription (RT-qPCR) assay. When combined with magnetic bead-based extraction, the high-throughput SHERLOCK SARS-CoV-2 assay was 100% concordant (n = 60) with the CDC RT-qPCR. When used with direct sample addition the high-throughput assay was also 100% concordant with the CDC RT-qPCR direct method (n = 45). With direct saliva sample addition, the negative and positive percentage agreements were 100% (15/15, 95% CI: 81.8-100%) and 88% (15/17, 95% CI: 63.6-98.5%), respectively, compared with results from a collaborating clinical laboratory. CONCLUSIONS: This high-throughput assay identifies SARS-CoV-2 from patient samples with or without nucleic acid extraction with high concordance to RT-qPCR methods. This test enables high complexity laboratories to rapidly increase their testing capacities with simple equipment.