Wastewater Surveillance for Identifying SARS-CoV-2 Infections in Long-Term Care Facilities, Kentucky, USA, 2021-2022.

Persons living in long-term care facilities (LTCFs) were disproportionately affected by COVID-19. We used wastewater surveillance to detect SARS-CoV-2 infection in this setting by collecting and testing 24-hour composite wastewater samples 2-4 times weekly at 6 LTCFs in Kentucky, USA, during March 2021-February 2022. The LTCFs routinely tested staff and symptomatic and exposed residents for SARS-CoV-2 using rapid antigen tests. Of 780 wastewater samples analyzed, 22% (n = 173) had detectable SARS-CoV-2 RNA. The LTCFs reported 161 positive (of 16,905) SARS-CoV-2 clinical tests. The wastewater SARS-CoV-2 signal showed variable correlation with clinical test data; we observed the strongest correlations in the LTCFs with the most positive clinical tests (n = 45 and n = 58). Wastewater surveillance was 48% sensitive and 80% specific in identifying SARS-CoV-2 infections found on clinical testing, which was limited by frequency, coverage, and rapid antigen test performance.

Simple SARS-CoV-2 concentration methods for wastewater surveillance in low resource settings.

Wastewater-based epidemiology (WBE) measures pathogens in wastewater to monitor infectious disease prevalence in communities. Due to the high dilution of pathogens in sewage, a concentration method is often required to achieve reliable biomarker signals. However, most of the current concentration methods rely on expensive equipment and labor-intensive processes, which limits the application of WBE in low-resource settings. Here, we compared the performance of four inexpensive and simple concentration methods to detect SARS-CoV-2 in wastewater samples: Solid Fraction, Porcine Gastric Mucin-conjugated Magnetic Beads, Calcium Flocculation-Citrate Dissolution (CFCD), and Nanotrap® Magnetic Beads (NMBs). The NMBs and CFCD methods yielded the highest concentration performance for SARS-CoV-2 (∼16-fold concentration and âˆ¼ 41 % recovery) and require <45 min processing time. CFCD has a relatively low consumable cost (<$2 per four sample replicates). All methods can be performed with basic laboratory equipment and minimal electricity usage which enables further application of WBE in remote areas and low resource settings.

Wastewater Surveillance for SARS-CoV-2 at Long-Term Care Facilities: Mixed Methods Evaluation.

BACKGROUND: Wastewater surveillance provided early indication of COVID-19 in US municipalities. Residents of long-term care facilities (LTCFs) experienced disproportionate morbidity and mortality early in the COVID-19 pandemic. We implemented LTCF building-level wastewater surveillance for SARS-CoV-2 at 6 facilities in Kentucky to provide early warning of SARS-CoV-2 in populations considered vulnerable. OBJECTIVE: This study aims to evaluate the performance of wastewater surveillance for SARS-CoV-2 at LTCFs in Kentucky. METHODS: We conducted a mixed methods evaluation of wastewater surveillance following Centers for Disease Control and Prevention (CDC) guidelines for evaluating public health surveillance systems. Evaluation steps in the CDC guidelines were engaging stakeholders, describing the surveillance system, focusing the evaluation design, gathering credible evidence, and generating conclusions and recommendations. We purposively recruited stakeholders for semistructured interviews and undertook thematic content analysis of interview data. We integrated wastewater, clinical testing, and process data to characterize or calculate 7 surveillance system performance attributes (simplicity, flexibility, data quality, sensitivity and positive predictive value [PPV], timeliness, representativeness, and stability). RESULTS: We conducted 8 stakeholder interviews. The surveillance system collected wastewater samples (N=811) 2 to 4 times weekly at 6 LTCFs in Kentucky from March 2021 to February 2022. Synthesis of credible evidence indicated variable surveillance performance. Regarding simplicity, surveillance implementation required moderate human resource and technical capacity. Regarding flexibility, the system efficiently adjusted surveillance frequency and demonstrated the ability to detect additional pathogens of interest. Regarding data quality, software identified errors in wastewater sample metadata entry (110/3120, 3.53% of fields), technicians identified polymerase chain reaction data issues (140/7734, 1.81% of reactions), and staff entered all data corrections into a log. Regarding sensitivity and PPV, using routine LTCF SARS-CoV-2 clinical testing results as the gold standard, a wastewater SARS-CoV-2 signal of >0 RNA copies/mL was 30.6% (95% CI 24.4%-36.8%) sensitive and 79.7% (95% CI 76.4%-82.9%) specific for a positive clinical test at the LTCF. The PPV of the wastewater signal was 34.8% (95% CI 27.9%-41.7%) at >0 RNA copies/mL and increased to 75% (95% CI 60%-90%) at >250 copies/mL. Regarding timeliness, stakeholders received surveillance data 24 to 72 hours after sample collection, with delayed reporting because of the lack of weekend laboratory staff. Regarding representativeness, stakeholders identified challenges delineating the population contributing to LTCF wastewater because of visitors, unknown staff toileting habits, and the use of adult briefs by some residents preventing their waste from entering the sewer system. Regarding stability, the reoccurring cost to conduct 1 day of wastewater surveillance at 1 facility was approximately US $144.50, which included transportation, labor, and materials expenses. CONCLUSIONS: The LTCF wastewater surveillance system demonstrated mixed performance per CDC criteria. Stakeholders found surveillance feasible and expressed optimism regarding its potential while also recognizing challenges in interpreting and acting on surveillance data.

Improving wastewater-based epidemiology performance through streamlined automation.

Wastewater-based epidemiology (WBE) has enabled us to describe Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections in populations. However, implementation of wastewater monitoring of SARS-CoV-2 is limited due to the need for expert staff, expensive equipment, and prolonged processing times. As WBE increases in scope (beyond SARS-CoV-2) and scale (beyond developed regions), there is a need to make WBE processes simpler, cheaper, and faster. We developed an automated workflow based on a simplified method termed exclusion-based sample preparation (ESP). Our automated workflow takes 40 min from raw wastewater to purified RNA, which is several times faster than conventional WBE methods. The total assay cost per sample/replicate is $6.50 which includes consumables and reagents for concentration, extraction, and RT-qPCR quantification. The assay complexity is reduced significantly, as extraction and concentration steps are integrated and automated. The high recovery efficiency of the automated assay (84.5 ± 25.4%) yielded an improved Limit of Detection (LoDAutomated=40 copies/mL) compared to the manual process (LoDManual=206 copies/mL), increasing analytical sensitivity. We validated the performance of the automated workflow by comparing it with the manual method using wastewater samples from several locations. The results from the two methods correlated strongly (r = 0.953), while the automated method was shown to be more precise. In 83% of the samples, the automated method showed lower variation between replicates, which is likely due to higher technical errors in the manual process e.g., pipetting. Our automated wastewater workflow can support the expansion of WBE in the fight against Coronavirus Disease of 2019 (COVID-19) and other epidemics.

Stabilization of SARS-CoV-2 RNA in wastewater via rapid RNA extraction.

Wastewater-based Epidemiology (WBE) has contributed to surveillance of SARS-CoV-2 in communities across the world. Both symptomatic and asymptomatic patients with COVID-19 can shed the virus through the gastrointestinal tract, enabling the quantification of the virus in stool and ultimately in wastewater (WW). Unfortunately, instability of SARS-CoV-2 RNA in wastewater limits the utility of WBE programs, particularly in remote/rural regions where reliable cold storage and/or rapid shipping may be unavailable. This study examined whether rapid SARS-CoV-2 RNA extraction on the day of sample collection could minimize degradation. Importantly, the extraction technology used in these experiments, termed exclusion-based sample preparation (ESP), is lightweight, portable, and electricity-free, making it suitable for implementation in remote settings. We demonstrated that immediate RNA extraction followed by ambient storage significantly increased the RNA half-life compared to raw wastewater samples stored at both 4 °C or ambient temperature. Given that RNA degradation negatively impacts both the sensitivity and precision of WBE measurements, efforts must be made to mitigate degradation in order to maximize the potential impact of WBE on public health.

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

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.