A multicenter study investigating SARS-CoV-2 in tertiary-care hospital wastewater. viral burden correlates with increasing hospitalized cases as well as hospital-associated transmissions and outbreaks

Abstract

SARS-CoV-2 has been detected in wastewater and its abundance correlated with community COVID-19 cases, hospitalizations and deaths. We sought to use wastewater-based detection of SARS-CoV-2 to assess the epidemiology of SARS-CoV-2 in hospitals. Between August and December 2020, twice-weekly wastewater samples from three tertiary-care hospitals (totaling > 2100 dedicated inpatient beds) were collected. Hospital-1 and Hospital-2 could be captured with a single sampling point whereas Hospital-3 required three separate monitoring sites. Wastewater samples were concentrated and cleaned using the 4S-silica column method and assessed for SARS-CoV-2 gene-targets (N1, N2 and E) and controls using RT-qPCR. Wastewater SARS-CoV-2 as measured by quantification cycle (Cq), genome copies and genomes normalized to the fecal biomarker PMMoV were compared to the total daily number of patients hospitalized with active COVID-19, confirmed cases of hospital-acquired infection, and the occurrence of unit-specific outbreaks. Of 165 wastewater samples collected, 159 (96%) were assayable. The N1-gene from SARS-CoV-2 was detected in 64.1% of samples, N2 in 49.7% and E in 10%. N1 and N2 in wastewater increased over time both in terms of the amount of detectable virus and the proportion of samples that were positive, consistent with increasing hospitalizations at those sites with single monitoring points (Pearson's r = 0.679, P < 0.0001, Pearson's r = 0.799, P < 0.0001, respectively). Despite increasing hospitalizations through the study period, nosocomial-acquired cases of COVID-19 (Pearson's r = 0.389, P < 0.001) and unit-specific outbreaks were discernable with significant increases in detectable SARS-CoV-2 N1-RNA (median 112 copies/ml) versus outbreak-free periods (0 copies/ml; P < 0.0001). Wastewater-based monitoring of SARS-CoV-2 represents a promising tool for SARS-CoV-2 passive surveillance and case identification, containment, and mitigation in acute- care medical facilities.

Keywords: COVID-19; Hospital-acquired; Sewage; Wastewater; Wastewater-based epidemiology.

Graphical abstract

Fig. 1

Determination of SARS-CoV-2 RNA in wastewater samples from Hospital-1 and Hospital-2. Relative SARS-CoV-2 genomic copies compared to genomic copies of PMMoV from (A) Hospital 1 (August 5^th to December 17^th) and (B) Hospital 2 (August 5^th to December 17^th). Quantification of SARS-CoV-2 RNA in samples was determined by the N1 (black) and N2 (red) assays. Green line denotes the total daily number of active prevalent cases in the hospital. Orange bars denotes the number of daily hospital-acquired cases. Plots show the average of three technical replicates and error bars represent the standard deviation. Vertical dash lines correspond to days where outbreaks were declared (Table S3), where the number of patients and health care workers involved are indicated at the top each dotted dash line. Asterisk denotes that for a specific outbreak more than one unit was involved. Gray zones denote duration of the outbreak. Bottom individual boxed areas represent individual samples as positive (+) samples where SARS-CoV-2 signal was identified with a Cq<40, and negatives (-) had values ≥40. Please note that the scale is different in Figures. A and B. HA: hospital acquired, HCW: health care worker.

Fig. 2

Determination of SARS-CoV-2 RNA in wastewater samples from Hospital-3. Relative SARS-CoV-2 genomic copies compared to genomic copies of PMMoV from (A) Hospital 3A (Trauma, Medical & Surgical ICUs, orthopedics surgery and designated COVID-care units) August 5^th to December 17^th), (B) Hospital 3B (i.e., Main Building, North wing, October 1^st to December 17^th) and (C) Hospital3C (i.e., Main Building South Wing, cancer care building, complex medical care building and hostel/administration building), October 1^st to December 17^th). Quantification of SARS-CoV-2 RNA in samples was determined by the N1 (black) and N2 (red) assays. Green line denotes the number of prevalent cases in the hospital. Orange bars denotes the number of daily hospital-acquired cases. Plots show the average of three technical replicates and error bars represent the standard deviation. Vertical dash lines correspond to days where outbreaks were declared (Table S3), where the number of patients and health care workers involved are indicated at the top each dotted dash line. Asterisk (*) denotes the largest outbreak which occurred initially at Hospital_3C prior to instituted monitoring at that site – and reflects the SARS-CoV-2 infected patients who were relocated to the designated COVID-19 wards in Hospital_3A where it was detected by wastewater monitoring. The last case associated with the large outbreak was identified October 19^th. Gray zones denote duration of the outbreak. Bottom individual boxed areas represent individual samples as positive (+) samples where SARS-CoV-2 signal was identified with a Cq<40, and negatives (-) had values ≥40. Please note that the scale is different from A, B and C Figures. HA: hospital acquired, HCW: health care worker.