Sars-Cov-2 Rna Concentrations in Primary Municipal Sewage Sludge As A Leading Indicator of Covid-19 Outbreak Dynamics
Sars-Cov-2 Rna Concentrations in Primary Municipal Sewage Sludge As A Leading Indicator of Covid-19 Outbreak Dynamics
Sars-Cov-2 Rna Concentrations in Primary Municipal Sewage Sludge As A Leading Indicator of Covid-19 Outbreak Dynamics
1
Department of Chemical and Environmental Engineering, School of Engineering and Applied
Science, Yale University, New Haven, CT, USA
2
Connecticut Agricultural Experimental Station, State of Connecticut, New Haven, CT, USA
3
Department of Epidemiology of Microbial Disease, School of Public Health, Yale University,
New Haven, CT, USA
4
School of Management, Yale University, New Haven, CT, USA
5
Yale School of Medicine, New Haven, CT, USA
6
Yale Institute for Global Health, New Haven, CT, USA
7
Yale School of Nursing, Orange, CT, USA
8
Public Health Modeling, Yale School of Public Health, New Haven, CT, USA
‡
Contributed equally to this work
*
Corresponding authors: Jordan Peccia, Tel: +1 (203) 432-4385; email: Jordan.Peccia@Yale.edu; Saad Omer, Tel
+1 (203) 432-3656; Saad.Omer@Yale.edu
medRxiv preprint doi: https://doi.org/10.1101/2020.05.19.20105999.this version posted May 22, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .
Abstract
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
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
Introduction
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
present4, 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 patients5-7 and has recently been
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
medRxiv preprint doi: https://doi.org/10.1101/2020.05.19.20105999.this version posted May 22, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .
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
Results
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 103 virus RNA copies mL-1 to 4.6 x 105
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
sludge matrix. Overall, 96.5% of all CT values were less than 38 and values between 38 and 40
were reported as positive if detection occurred with virus nucleocapsid N1 and N2 primer sets
and both replicates. Replicated samples demonstrated similar SARS-CoV-2 RNA concentration
values and comparisons between SARS-CoV-2 primer values with the human ribonuclease P
(RP) gene primer values indicate that dynamic temporal concentration changes in SARS-CoV-2
RNA were from actual change in virus concentration (Figure 1B). Concentration comparisons
medRxiv preprint doi: https://doi.org/10.1101/2020.05.19.20105999.this version posted May 22, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .
between replicates produced an average slope of 0.98 ± 0.17 st. dev., while replicates comparing
N and RP primer values result in a flat line, with average slope of 0.05 ± 0.05 st. dev. (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,B) and COVID-19 compiled testing data for the four
municipalities (New Haven, East Haven, Hamden, and Woodbridge, CT) served by the
ESWPAF (Figure 2C). All three measures traced the initial wave of the SARS-CoV-2 outbreak
in the New Haven metropolitan area. Applying Locally Weighted Scatterplot Smoothing
(LOWESS) to the data and rescaling enables comparison (Figure 2A,B). The virus RNA time
course peaked 3 days earlier than hospital admissions (April 9 versus April 12) and a cross
correlation analysis revealed a correlation coefficient (R=0.996) between smoothed RNA and
Comparing the LOWESS-smoothed daily virus RNA and COVID-19 testing data reveals that
the peak in COVID-19 cases (April 16) occurred 7 days following the peak in SARS-CoV-2
RNA concentration (April 9) (Figure 2C). A cross correlation analysis resulted in a maximum
correlation coefficient (R=0.994) when the COVID-19 testing data is adjusted 7 days forward.
Regressing the adjusted COVID-19 testing data and SARS-CoV-2 virus RNA concentrations
yield a value of 1,305 virus RNA copies/mL per new COVID-19 case report as the outbreak
ascended (Figure 2D), and 1,240 virus RNA copies/mL per new COVID-19 case report as the
Discussion
during a COVID-19 outbreak in the New Haven, CT metropolitan area. Approximately 200,000
people are served by the treatment facility and COVID-19 total documented cases by testing rose
from less than 29 to 2,609 during the March 19 to May 1, 2020 surveillance period. Our results
demonstrate: (1) the utility of SARS-CoV-2 primary sludge monitoring to accurately track
outbreaks in a community and (2) primary sludge SARS-CoV-2 RNA concentrations can be a
leading indicator over other commonly used epidemiology approaches including summarized
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 reported8, 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,
outbreak dynamics over hospitalization and compiled COVID-19 testing data. SARS-CoV-2
RNA concentrations led hospital admissions by 3 days and COVID-19 cases by 7 days. Hospital
admissions to Yale New Haven Hospital from the four towns served by the wastewater treatment
facility both rose and fell more slowly than the observed RNA concentrations. The rates of
SARS-CoV-2 RNA increases and decreases were similar to the COVID-19 testing data, and
reflect the very concentrated outbreak that took place within New Haven and its neighboring
towns. The strong correlation between the virus RNA and COVID-19 case data provides an
approach to independently evaluate the local SARS-CoV-2 testing rate and to estimate the
number of new cases. While the results here demonstrate the potential utility of such an
approach, we note that these relationships could be treatment plant specific, as primary sludge
process trains are not uniform, and that compiled COVID-19 testing is prompted by symptoms,
In conclusion, the SARS-CoV-2 RNA concentrations in primary sewage sludge were tracked
epidemiology curves established by compiled COVID-19 testing data and hospital admissions,
but was a leading indicator by seven and three days, respectively. Our study could have
easing restrictions, especially when there are limitations in clinical testing. Raw wastewater and
sludge-based surveillance is particularly useful for low and middle-income countries where
Methods
Sample collection. Primary sewage sludge was collected from the East Shore Water Pollution
Abatement Facility (ESWPAF) in New Haven, CT, USA. Samples were taken daily from March
19 to May 1, 2020 between 8 and 10 am EDT and stored at -80oC prior to analysis. The first
sampling dates were prior to widespread individual testing in the region, and prior to the start of
stay at home restrictions implemented throughout the State of Connecticut, USA (Figure 1A).
From the sampling start and end dates, cities served by the ESWPAF experienced greater than 85
times increase in confirmed COVID-19 cases (by testing) from less than 29 cases to 2,609.17 The
plant serves an estimated population of 200,000 people with average treated flows of 1.75 m3/s.
Sludge collected from ESWPAF is primary sludge, sampled at the outlet of a gravity thickener,
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
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
medRxiv preprint doi: https://doi.org/10.1101/2020.05.19.20105999.this version posted May 22, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .
followed by 40 cycles of 95°C for 10 s and 60°C for 30 s per the manufacturer’s
previously described18 and presented as virus RNA copies. The SARS-CoV-2 concentration
results were normalized to the total RNA extracted to reduce day to day variations in treatment
plant flow, sludge solids content, and RNA extraction efficiency. All samples were diluted 5x for
use as template to ensure the removal of inhibition. Performing qRT-PCR on undiluted and 5x
diluted samples typically resulted in the expected 5x decrease in concentration. Sewage sludge
from 2018 was used as a control and no SARS-CoV-2 detection was observed from either N1 or
N2 primers. These control sludges were consistently positive for the human RP gene.
Epidemiological Analysis. Daily COVID-19 admissions to the Yale New Haven Hospital
were compiled from hospital records and confirmed by laboratory testing. Numbers of
laboratory-confirmed COVID-19 cases in the towns served by the ESWPAF (New Haven, East
Haven, Hamden, and Woodbridge CT) were compiled from daily reports published by the CT
Department of Public Health.17 SARS-CoV-2 RNA, COVID-19 cases, and hospital admissions
time series were smoothed using Locally Weighted Scatterplot Smoothing (LOWESS) to better
Acknowledgements
We wish to thank the East Shore Water Pollution Abatement Facility, New Haven, CT, USA
for primary sludge sampling assistance. AZ is supported by the Yale Institute of Global Health
and NDG is supported by a gift from the Huffman Family Donor Advised Fund.
medRxiv preprint doi: https://doi.org/10.1101/2020.05.19.20105999.this version posted May 22, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .
References
1. Dong, E.; Du, H.; Gardner, L., An interactive web-based dashboard to track COVID-19
in real time. Lancet Infect Dis 2020, 20 (5), 533-534.
2. Mizumoto, K.; Kagaya, K.; Zarebski, A.; Chowell, G., Estimating the asymptomatic
proportion of coronavirus disease 2019 (COVID-19) cases on board the Diamond
Princess cruise ship, Yokohama, Japan, 2020. Euro Surveill 2020, 25 (10), 2000180.
3. García-Basteiro, A. L.; Chaccour, C.; Guinovart, C.; Llupià, A.; Brew, J.; Trilla, A.;
Plasencia, A., Monitoring the COVID-19 epidemic in the context of widespread local
transmission. Lancet Respir Med 2020, 8 (5), 440-442.
4. He, X.; Lau, E. H. Y.; Wu, P.; Deng, X.; Wang, J.; Hao, X.; Lau, Y. C.; Wong, J. Y.;
Guan, Y.; Tan, X.; Mo, X.; Chen, Y.; Liao, B.; Chen, W.; Hu, F.; Zhang, Q.; Zhong,
M.; Wu, Y.; Zhao, L.; Zhang, F.; Cowling, B. J.; Li, F.; Leung, G. M., Temporal
dynamics in viral shedding and transmissibility of COVID-19. Nat Med 2020, 26 (5),
672-675.
5. Wang, W.; Xu, Y.; Gao, R.; Lu, R.; Han, K.; Wu, G.; Tan, W., Detection of SARS-
CoV-2 in different types of clinical specimens. JAMA 2020, 323 (18), 1843-1844.
6. Zhang, J.; Wang, S.; Xue, Y., Fecal specimen diagnosis 2019 novel coronavirus-infected
pneumonia. J Med Virol 2020, doi: 10.1002/jmv.25742.
7. Xu, Y.; Li, X.; Zhu, B.; Liang, H.; Fang, C.; Gong, Y.; Guo, Q.; Sun, X.; Zhao, D.;
Shen, J.; Zhang, H.; Liu, H.; Xia, H.; Tang, J.; Zhang, K.; Gong, S., Characteristics of
pediatric SARS-CoV-2 infection and potential evidence for persistent fecal viral
shedding. Nat Med 2020, 26 (4), 502-505.
8. Ahmed, W.; Angel, N.; Edson, J.; Bibby, K.; Bivins, A.; O'Brien, J. W.; Choi, P. M.;
Kitajima, M.; Simpson, S. L.; Li, J.; Tscharke, B.; Verhagen, R.; Smith, W. J. M.;
Zaugg, J.; Dierens, L.; Hugenholtz, P.; Thomas, K. V.; Mueller, J. F., First confirmed
detection of SARS-CoV-2 in untreated wastewater in Australia: A proof of concept for
the wastewater surveillance of COVID-19 in the community. Science of The Total
Environment 2020, 728, 138764.
9. Medema, G.; Heijnen, L.; Elsinga, G.; Italiaander, R.; Brouwer, A., Presence of SARS-
Coronavirus-2 in sewage. medRxiv 2020, 2020.03.29.20045880.
10. Wu, F.; Xiao, A.; Zhang, J.; Gu, X.; Lee, W. L.; Kauffman, K.; Hanage, W.; Matus,
M.; Ghaeli, N.; Endo, N.; Duvallet, C.; Moniz, K.; Erickson, T.; Chai, P.; Thompson,
J.; Alm, E., SARS-CoV-2 titers in wastewater are higher than expected from clinically
confirmed cases. medRxiv 2020, 2020.04.05.20051540.
11. Pöyry, T.; Stenvik, M.; Hovi, T., Viruses in sewage waters during and after a
poliomyelitis outbreak and subsequent nationwide oral poliovirus vaccination campaign
in Finland. Appl Environ Microbiol 1988, 54 (2), 371.
12. Bibby, K.; Peccia, J., Identification of viral pathogen diversity in sewage sludge by
metagenome analysis. Environ Sci Technol 2013, 47 (4), 1945-1951.
medRxiv preprint doi: https://doi.org/10.1101/2020.05.19.20105999.this version posted May 22, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .
13. Wurtzer, S.; Marechal, V.; Mouchel, J.-M.; Maday, Y.; Teyssou, R.; Richard, E.;
Almayrac, J. L.; Moulin, L., Evaluation of lockdown impact on SARS-CoV-2 dynamics
through viral genome quantification in Paris wastewaters. medRxiv 2020,
2020.04.12.20062679.
14. Zhang, Q. H.; Yang, W. N.; Ngo, H. H.; Guo, W. S.; Jin, P. K.; Dzakpasu, M.; Yang,
S. J.; Wang, Q.; Wang, X. C.; Ao, D., Current status of urban wastewater treatment
plants in China. Environ Int 2016, 92-93, 11-22.
15. U.S. Cybersecurity and Cyber Intrastructure. Water and Wastewater Systems Sector:
Critical Infrastructure Sectors. Washington, D.C.: U.S. Department of Homeland
Security. 2016-01-08. 2018.
16. Li, R.; Pei, S.; Chen, B.; Song, Y.; Zhang, T.; Yang, W.; Shaman, J., Substantial
undocumented infection facilitates the rapid dissemination of novel coronavirus (SARS-
CoV-2). Science 2020, 368 (6490), 489.
17. State of Connecticut, CT Data: Confirmed COVID-19 Cases. Connecticut Department of
Health, https://data.ct.gov/Health-and-Human-Services/COVID-19-confirmed-cases-by-
town-/28fr-iqnx Access, 5/3/2020. 2020.
18. Vogels, C. B. F.; Brito, A. F.; Wyllie, A. L.; Fauver, J. R.; Ott, I. M.; Kalinich, C. C.;
Petrone, M. E.; Casanovas-Massana, A.; Muenker, M. C.; Moore, A. J.; Klein, J.; Lu,
P.; Lu-Culligan, A.; Jiang, X.; Kim, D. J.; Kudo, E.; Mao, T.; Moriyama, M.; Oh, J.
E.; Park, A.; Silva, J.; Song, E.; Takehashi, T.; Taura, M.; Tokuyama, M.;
Venkataraman, A.; Weizman, O.-E.; Wong, P.; Yang, Y.; Cheemarla, N. R.; White,
E.; Lapidus, S.; Earnest, R.; Geng, B.; Vijayakumar, P.; Odio, C.; Fournier, J.;
Bermejo, S.; Farhadian, S.; Dela Cruz, C.; Iwasaki, A.; Ko, A. I.; Landry, M.-L.;
Foxman, E. F.; Grubaugh, N. D., Analytical sensitivity and efficiency comparisons of
SARS-COV-2 qRT-PCR primer-probe sets. medRxiv 2020, 2020.03.30.20048108.
19. Cleveland, W. S., Robust locally weighted regression and smoothing scatterplots. Journal
of the American Statistical Association 1979, 74 (368), 829-836.
medRxiv preprint doi: https://doi.org/10.1101/2020.05.19.20105999.this version posted May 22, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .
Figures
500,000
A B
400,000
Stay at Home Rep 2 RP
23-Mar
100 300,000
Slope=0.97
200,000
50
100,000
Slope=-0.008
0 0
0 200000 400000
19-Mar 29-Mar 8-Apr 18-Apr 28-Apr
viral RNA/mL Rep1 (N1)
500,000
C Rep 1-N1 Rep 1-N2 Rep 2-N1 Rep 2-N2
400,000
viral RNA/mL
300,000
200,000
100,000
0
19-3 1-5 19-3 1-5 19-3 1-5 19-3 1-5
Figure 1. (A) Epidemiology curve for COVID-19 new cases (based on testing) from the
cities in the wastewater catchment area (New Haven, East Haven, and Hamden, CT)
served by the East Shore Water Pollution Abatement Facility (ESWPAF)1; (B) Example
comparison of SARS-CoV-2 virus RNA replicates (N1 primer set) and comparison
between N1 primer concentrations versus human RP concentrations; (C) SARS-CoV-2
virus RNA concentration time course for all replicates and primers considered.
medRxiv preprint doi: https://doi.org/10.1101/2020.05.19.20105999.this version posted May 22, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .
400,000 40
A 150,000
Hospital Admissions
Hospital Admissions
B
virus RNA/mL
(Patients/Day)
virus RNA/mL
(Patients/Day)
300,000 30 20
100,000
200,000 20
50,000 10
100,000 10
0 0 0 0
19-Mar 2-Apr 16-Apr 30-Apr 19-Mar 2-Apr 16-Apr 30-Apr
150,000
C 150,000
100,000 100,000
100,000
50
50,000 50,000
75,000
Slope= 1,240 Slope= 1,305
0 0 0
19-Mar 2-Apr 16-Apr 30-Apr 0 50 100 50 75 100
New COVID-19 Cases New COVID-19 Cases
Figure 2. (A) Average sludge SARS-CoV-2 RNA concentration time course data (o)
and average hospital admissions data (o) with LOWESS smoothing; (B) rescaled
smoothed SARS-CoV-2 virus RNA concentrations (---) and hospital admissions (---);
(C) smoothed sludge SARS-CoV-2 virus RNA concentration (---) with smoothed COVID-
19 epidemiology curve (---); (D) regression between smoothed virus RNA and new
COVID-19 cases (ascending), slope=1,240 virus RNA copies/new case, R2=0.99; (E)
regression between smoothed virus RNA and new COVID-19 cases (descending),
slope=1,305 virus RNA copies/new case, R2=0.97.