Quantification of SARS-CoV-2 and cross-assembly phage (crAssphage) from wastewater to monitor coronavirus transmission within communities

Abstract

Wastewater surveillance of SARS-CoV-2 has become an attractive tool for combating the spread of COVID-19 by assessing the presence or levels of the virus shed in a population. However, the methods to quantify viral RNA and to link those quantities to the level of infection within the community vary. In this study, we sought to identify and optimize scalable methods for recovery of viral nucleic acids from wastewater and attempted to use a constitutive member of the gut virome, human-specific crAssphage, to help account for unknown levels of SARS-CoV-2 decay and dilution in the wastewater infrastructure. Results suggest that ultracentrifugation of a small volume of wastewater through a 50% sucrose cushion followed by total nucleic acid extraction yielded quantifiable virus in an area with a modest number of COVID-19 cases. Further, the ratio of log10(SARS-CoV-2):log10(crAssphage) appears to be associated with the cumulative incidence of COVID-19 in the Syracuse, NY area. In areas where ultracentrifuges are available, these methods may be used to link SARS-CoV-2 quantities in wastewater to levels of transmission within communities with sewer service.

Sewage analysis as a tool for the COVID-19 pandemic response and management: the urgent need for optimised protocols for SARS-CoV-2 detection and quantification

“…To this end, even though more information is needed on the exact aspects of transmission of the alive virus through faeces via the oral-faecal pathway and of its infectivity in sewage, the presence of SARS-CoV-2 RNA in wastewater was described worldwide; in the Netherlands ([ 21 ] [ preprint] ; [ 38 ]), Italy ([ [22] , [23] ]; [ 24 ] [ preprint] ; [ 9 ] [ preprint] ), France ([ 25 ] [ preprint] ; [ 6 ] [ preprint] ), Spain [ 26 ], Israel ([ 27 ] [ preprint] ), Turkey ([ 28 ] [ preprint] ), USA ([ 29 ] [ preprint] ; [ 8 ] [ preprint] ; [ 30 ]; [ 31 ] [ preprint] ), India ([ 32 ] [ preprint] ), Japan ([ 33 ] [ preprint] ), Brazil ([ 7 ] [ preprint] ) and Australia [ 4 , 34 , 35 ].…”

“…The studies reviewed herein, dedicated to the detection of SARS-CoV-2 in urban wastewater, were published between the 30 th of March 2020 to the 15 th of July 2020, with the majority of them not being certified by formal peer review. More specifically, at the time of preparing this review, thirteen (13) out of the twenty (20) studies were published in the literature as preprints ([ 27 ] [ preprint] ; [ 7 ] [ preprint] ; [ 29 ] [ preprint] ; [ 33 ] [ preprint] ; [ 28 ] [ preprint] ; [ 32 ] [ preprint] ; [ 21 ] [ preprint] ; [ 8 ] [ preprint] ; [ 9 ] [ preprint] ; [ 25 ] [ preprint] ; [ 31 ] [ preprint] ; [ 6 ] [ preprint] ; [ 24 ] [ preprint ]). It is clear that there is an increased scientific and public engagement with the COVID-19 preprints.…”

“…), 15 th , 16 th and 25 th of March, 2020 (1135, 1413, and 6412 reported COVID-19 cases, resp.) 24 h flow-dependent composite wastewater sample (250 mL) not reported freezing of samples at 4 °C andtransfer on melting ice not reported not reported × [ 28 ]* Turkey 7 UWTPs in Istanbul, Turkey and 2 manholes nearby Istanbul hospitals Preliminary treatment; CAS; advanced nutrient removal 21 st and 25 th of April, 2020 grab samples 250-mL sterilized bottles not reported not reported not reported × [ 29 ]* USA 11 points of sampling (i.e., 6 wastewater treatment plants, and 5 inlet pumping stations, or interceptor lines) in Syracuse, NY and other locations in Onondaga County, NY. PE = 12800-223900 (UWTPs) PE = 3841-112747 (interceptor lines) 6 th and 13 th May, 2020 24 hour-composite wastewater samples not reported storage of samples at 4 °C andtransportation on ice to Upstate Medical University (Syracuse, NY) not reported Next morning × [ 8 ]* USA UWTP in Bozeman, Montana 18 th -25 th of May, 2020 Collection of 0.5-L samples of pre-treated wastewater on 7 days within a 17-day period, using two collection methods (grab and 24 h-composite samples) not reported not reported not reported not reported × [ 30 ] USA 9 composite and 6 grab wastewater samples from two UWTPs in Southern Louisiana, respectively (UWTP A = 244627 PE; UWTP B = 45694 PE) 7 raw wastewater samples 4 secon...…”

“…15 mL centrifugation (2000×g, 10 min) at 4 °C filtration (0.45 μm pore size, diameter 25 mm) of the supernatant and concentration using a Centriprep YM-50 ultrafiltration device (ii) adsorption-direct RNA extraction : filtration of the water samples with 25 mM MgCl2 through a mixed cellulose-ester membrane (pore size 0.8 μm; diameter 90 mm) - - [ 26 ] Spain inoculation of bio-banked influent (200 mL) and effluent wastewater samples with PEDV and MgV pH adjustment to 6.0 and addition of 0.9 N of AlCl3 (1:100) readjustment of pH to 6.0 and mixing with an orbital shaker (150 rpm, 15 min) centrifugation (1700×g, 20 min) re-suspension of pellet in 10 mL of 3% beef extract (pH 7.4) mixing of the samples (150 rpm, 10 min) centrifugation (1900×g, 30 min) pellet resuspension in 1 mL of PBS PEDV strain CV777 MgV influent: PEDV = 11 ± 2.1% MgV = 11 ± 3.5% effluent: PEDV = 3.3 ± 1.6% MgV = 6.2 ± 1.0% [ 21 ]* [ 38 ] The Netherlands centrifugation (4654×g, 30 min) to remove large particles filtration of 100-200 mL supernatant with Centricon® Plus-70 centrifugal ultrafilter (10 kDa cut-off) by centrifugation (1500×g, 15 min) F-specific RNA 73 ± 50% [ 28 ]* Turkey centrifugation (3200×g, 45 min) of 250 mL of wastewater influent for removal of large particles filtration of 250 mL of supernatant with Amicon® Ultra-15 (10 kDa cut-off) by centrifugation (3200×g, 25-40 min) collection of 200-600 μL of concentrate - - [ 29 ]* USA mixing of samples to re-suspend particulates 20 mL was aliquoted to a 38.5 mL ultracentrifuge tube addition of a 12 mL sucrose cushion (50% sucrose in TNE buffer [20 mM Tris-HCl (pH 7.0), 100 mM NaCl, 2 mM EDTA]) purification of samples by centrifugation (150000×g, 90 and 45 min) at 4 °C using a Sorvall WX Ultra series wit...…”

“…Both grab ( [8][preprint] ;[ 34 ]; [9][preprint] ; [ 26 ]) and composite( [27][preprint] ; [29][preprint] ; [21][preprint] ;[ 23 , 38 ]; [8][preprint] ; [31][preprint] ) sampling methods have been reported for the collection of wastewater samples for the detection of SARS-CoV-2. In the case of composite samples, both time- (i.e.…”