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.

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.