key: cord-354051-ro3o27pv authors: Peccia, J.; Zulli, A.; Brackney, D. E.; Grubaugh, N. D.; Kaplan, E. H.; Casanovas-Massana, A.; Ko, A. I.; Malik, A. A.; Wang, D.; Wang, M.; Weinberger, D. M.; Omer, S. B. title: SARS-CoV-2 RNA concentrations in primary municipal sewage sludge as a leading indicator of COVID-19 outbreak dynamics date: 2020-05-22 journal: nan DOI: 10.1101/2020.05.19.20105999 sha: doc_id: 354051 cord_uid: ro3o27pv We report a time course of SARS-CoV-2 RNA concentrations in primary sewage sludge during the Spring COVID-19 outbreak in a northeastern U.S. metropolitan area. SARS-CoV-2 RNA was detected in all environmental samples and, when adjusted for the time lag, the virus RNA concentrations were highly correlated with the COVID-19 epidemiological curve (R2=0.99) and local hospital admissions (R2=0.99). SARS-CoV-2 RNA concentrations were a seven-day leading indicator ahead of compiled COVID-19 testing data and led local hospital admissions data by three days. Decisions to implement or relax public health measures and restrictions require timely information on outbreak dynamics in a community. The most common metric followed to track the progression of the COVID-19 epidemic within communities is derived from testing symptomatic cases and evaluating the number of positive tests over time. 1 However, tracking positive tests is a lagging indicator for the epidemic progression. 2, 3 Testing is largely prompted by symptoms, which may take up to five days to present 4 , and individuals can shed virus prior to exhibiting symptoms. There is a pressing need for additional methods for early sentinel surveillance and real-time estimations of community disease burden so that public health authorities may modulate and plan epidemic responses accordingly. SARS-CoV-2 RNA is present in the stool of COVID-19 patients [5] [6] [7] and has recently been documented in raw wastewater. [8] [9] [10] Thus, monitoring raw wastewater (sewage) within a community's collection system can potentially provide information on the prevalence and dynamics of infection for entire populations. 11 When municipal raw wastewater discharges into treatment facilities, solids are settled and collected into a matrix called (primary) sewage sludge, which has been shown to contain a broad diversity of human viruses including commonly circulating coronavirus strains. 12 Primary sludge provides a well-mixed and concentrated sample that may be advantageous for monitoring SARS-CoV-2. As viral shedding can occur before cases are detected, we hypothesize that the time course of SARS-CoV-2 RNA concentrations in primary sewage sludge is a leading indicator of outbreak dynamics within a community served by the treatment plant. During the COVID-19 outbreak from March 19, 2020 to May 1, 2020 in the New Haven, Connecticut (CT), USA metropolitan area ( Figure 1A) , daily primary sludge samples were collected from the wastewater treatment facility which serves approximately 200,000 residents. SARS-CoV-2 viral RNA concentrations were quantitatively compared with local hospital admission data and community COVID-19 compiled testing data. SARS-CoV-2 viral RNA was detectable in all samples tested and ranged from 1.7 x 10 3 virus RNA copies mL -1 to 4.6 x 10 5 virus RNA copies mL -1 . The lower concentration in this range corresponds to a qRT-PCR cycle threshold (CT) value of 38.75 and can be considered a detection threshold for this method and Figure 1B) . Overall, the SARS-CoV-2 RNA concentration time series results consistently reveal a curve that increases over times, peaks, and then decreases to May 1 ( Figure 1C ). New Haven COVID-19 epidemic suggest that these data may provide useful epidemiological insights ( Figure 1C ). SARS-CoV-2 RNA sludge concentrations were quantitatively compared with data that are commonly used to track the community progression of COVID-19 including hospital admissions (Figure 2A This study uniquely utilized primary sewage sludge instead of raw wastewater for virus RNA measurements. Sewage sludge is comprised of solids that are removed during primary sedimentation steps and typically gravity thickened. As a result, primary sludge has a greater solids content (2 to 5%) than raw wastewater (0.01 to 0.05%). Due to the elevated solids content and the high case load observed during the outbreak (~1,200 per 100,000 population), the concentrations of SARS-CoV-2 RNA reported here ranged from two to three orders of magnitude greater than raw wastewater SARS-CoV-2 values previously reported 8, 10, 13 . Monitoring primary sludge is broadly applicable. Wastewater treatment plants with primary and secondary treatment are standard in many regions of the world, and treatment facilities are rapidly expanding in urban areas of lower and middle-income countries. 14 Within the US, approximately 16,000 treatment plants serve more than 250,000,000 people. 15 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Viral RNA quantitative testing. To quantify SARS-CoV-2 virus RNA concentrations in primary sludge, 2.5 mL of well mixed sludge were added directly to a commercial kit optimized for isolation of total RNA from soil (RNeasey PowerSoil Total RNA kit, Qiagen). Isolated RNA pellets were dissolved in 50 μL of ribonuclease free water and total RNA measured by spectrophotometry (NanoDrop, ThermoFisher Scientific). SARS-CoV-2 RNA was quantified by one-step quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) using the U.S. Center for Disease Control N1 and N2 primers sets. 18 For control, analysis was also conducted for the human Ribonuclease P (RP) gene. Samples were analyzed using the Bio-Rad iTaq Universal Probes One-Step kit in 20 µL reactions run at 50°C for 10 min, 95°C for 1 min . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 22, 2020. . https://doi.org/10.1101/2020.05.19.20105999 doi: medRxiv preprint followed by 40 cycles of 95°C for 10 s and 60°C for 30 s per the manufacturer's recommendations. SARS-CoV-2 RNA concentrations were determined using a standard curve as previously described 18 is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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