key: cord-0704614-74ax8gx0
authors: Zhou, Nicolette A.; Tharpe, Courtney; Meschke, John Scott; Ferguson, Christobel
title: Survey of rapid development of environmental surveillance methods for SARS-CoV-2 detection in wastewater
date: 2021-01-14
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2020.144852
sha: a59ed0f0bf6bf509ad5098c60fe609e6ffdf19e6
doc_id: 704614
cord_uid: 74ax8gx0

Environmental surveillance as a part of wastewater-based epidemiology (WBE) of SARS-CoV-2 can provide an early, cost-effective, unbiased community-level indicator of circulating COVID-19 in a population. The objective of this study was to determine how widely SARS-CoV-2 detection in wastewater is being investigated and what methods are used. A survey was developed and distributed, with results showing that methods were rapidly applied to conduct SARS-CoV-2 WBE, primarily to test wastewater influent from large urban wastewater treatment plants. Additionally, most methods utilized small wastewater volumes and the primary concentration methods used were polyethylene glycol precipitation, membrane filtration and centrifugal ultrafiltration followed by nucleic acid extraction and assay for primarily nucleocapsid gene targets (N1, N2, and/or N3). Since this survey was performed, many laboratories have continued to optimize and implement a variety of methods for SARS-CoV-2 WBE. Method comparison studies completed since this survey was conducted will assist in developing WBE as a supplemental tool to support public health and policy decision making responses.

SARS-CoV-2 is the enveloped virus responsible for the COVID-19 pandemic, infecting more than 76 million people and resulting in the death of over 1.6 million globally as of December 21, 2020 1 . The virus belongs to the family Coronaviridae and genus betacoronavirus. SARS-CoV-2 is approximately 100-130 nm in size with a 30 kb positive sense RNA genome. The primary transmission route for the virus is via respiratory droplets 2 , though recent evidence has also shown that the virus infects cells in the gastrointestinal tract and is readily shed in stool 3, 4 .

Environmental surveillance is the monitoring of community health indicators of interest by periodically collecting and analyzing wastewater samples from sewers for the presence of chemical or microbiological targets. Environmental surveillance of wastewater is not a new area of research, and is now defined as part of wastewater-based epidemiology (WBE) 5 . It has been critically important in detecting the presence of wild and vaccine strains of poliovirus to support the World Health Organization (WHO) program of eradication of poliomyelitis 6, 7 and has also been used to investigate opioid use in communities 8 . What is new, however, is the exploration of its potential to provide an early, cost-effective, unbiased, integrated, community-level indication of (trends in) the presence of COVID-19 9-12 . As the pandemic unfolded the water sector rapidly engaged and responded to the crisis, seeking to increase knowledge and understanding of the virus and its potential pathways for infection. Early studies indicated that RNA indicative of the SARS-CoV-2 virus could be detected in wastewater and potentially serve as an early warning of infection levels in a community [13] [14] [15] [16] [17] [18] [19] [20] [21] Results from a survey focused on WBE methods for SARS-CoV-2 are described here, representing a snapshot of how widely the methods are being investigated in the water sector and what methodological approaches are being used. This survey was conducted as SARS-CoV-2 is a novel enveloped viral pathogen, potentially requiring use of different sampling and processing techniques than routinely used for other viral pathogens and this has been explored by method comparison studies [22] [23] [24] . Knowledge sharing and coordination of effort is essential to accelerate development of effective methods for SARS-CoV-2 detection and support decision making and policy responses to COVID-19.

The Water Research Foundation (WRF) released a 42 question survey via social media to collect information on the development of methods for the detection of genes indicating the presence of SARS-CoV-2 in wastewater (Appendix A). The survey was distributed via targeted emails, professional organizations, LinkedIn, Instagram, Twitter and Facebook and was open from the 16 th to 24 th April 2020.

One-hundred and sixty-nine (169) responses to this survey were received. The responses were reviewed to remove replicate responses based on replicate IP addresses (22) , affiliations (13) , and entries (7). This resulted in 127 responses. Of these, 32 (25.2%) respondents answered all of the questions and 95 (74.8%) provided partial responses.

J o u r n a l P r e -p r o o f Responses were received from 35 countries with the highest response from the United States (50), followed by Australia (8) , Canada (7), Japan (7) and India (5) ( Table 1) .

The survey was targeted and distributed to the water industry and responses came from a broad representation of organizations that contribute to this sector including academia (52.0%), industry and utilities (27.3%), state and federal government agencies (15.6%) and other groups including individuals, not-for-profit groups and consultants (11.7%). Respondents to the survey were mostly water industry individuals with technical expertise and identified their roles as either scientific, technical, operations or management responsibility suggesting they have familiarity with these methods and techniques (see Figure 1 ).

Although the survey was conducted in mid-April 2020, more than half of the respondents 54.3% (38/70) were already sampling and testing wastewater for the presence of SARS-CoV-2 (42.9%) or other targets of interest (11.4%) for the purpose of WBE, and the other 45.7% (32/70) indicated that they planned to commence environmental surveillance as soon as they had methods ready.

The questions respondents were aiming to address with WBE and what they were going to use the data for widely varied with focus on compliance/policy (relaxation of social distancing measures), methods comparison and method optimization. Many focused on research questions about what could be learnt about SARS-CoV-2 using environmental surveillance to determine the relationship between genomic copies and prevalence estimations, to track temporal patterns of SARS-CoV-2 in wastewater, to determine if additional waves of infections can be predicted, and to inform models.

J o u r n a l P r e -p r o o f Journal Pre-proof A question was posed to understand the types of locations sampled (question 7: urban, peri urban, rural, or other), indicating the majority of respondents already conducting environmental surveillance focused on urban sites (93.9%) with relatively few respondents additionally or solely sampling or monitoring from peri urban (28.6%) or rural (12.2%) sites. The populations served by the urban locations tended to be very large, with 87.0% of respondents indicating samples were from sites with catchment areas with more than 50,000 (56.8%) and/or 500,000 (56.8%) people. Less than 10% of sampling was targeted to areas with <500 people. When this survey was conducted, COVID-19 incidence was highest in urban areas.

This has changed in recent months with peri urban and rural areas now showing the greatest incidence of COVID-19 25 , suggesting increased WBE in rural areas may be needed.

With the rapid spread of SARS-CoV-2 and incubation period of 2-14 days 2 , sampling frequency and type (grab vs. composite) is important for monitoring trends in virus circulation and type. Therefore, the survey sought to gain information on sampling frequency with most respondents (44.7%) indicating that they were sampling wastewater weekly. While 36.2% indicated they were sampling either fortnightly or monthly. The majority of samples collected were taken as grab samples (63.8%) and/or composite samples (72.3%). Where composite samples were taken, they were predominantly representative of a 24 hour time period. Additionally, limited information is available on the persistence of this virus, therefore its survival in different matrices may vary making understanding of the matrices tested important.

Although a variety were monitored including at various stages of effluent treatment, the majority of respondents indicated they were sampling raw sewage at the influent to the wastewater plant (85.4%).

The next most tested matrices were secondary effluent (41.7%) and primary effluent (22.9%). Sampling at pumping stations within the sewer network represented 14.6% of responses. Other types of samples were also being tested by 33.3% of respondents including; primary solids, sludge, disinfected effluent and river water. Finally, the volume of sample being collected for analysis varied widely from as little as 50 J o u r n a l P r e -p r o o f mL up to as large as 100 L with larger sample volumes being applicable to treated effluents and cleaner water matrices and the smaller volumes typically being used for influent and primary effluent.

Virus survival could be affected by transportation and storage conditions and duration. As such, information was gathered about conditions used to assist future researchers. Of the 47 responses to question 17 about shipping conditions and time, a majority shipped samples on ice or with cold packs (36; 76.6%). Refrigeration was also infrequently used (3; 6.4%). A few respondents noted that shipping conditions were not applicable (7; 14.9%); it is unknown if this is due to samples not being shipped or not being transported on cold chain. Most samples were transported back to the laboratory in less than 4 hours 

Processing of samples for SARS-CoV-2 detection involves multiples steps such as preliminary treatment, primary concentration, secondary concentration, purification, extraction and/or detection (Figure 2 ). Not all steps are used for every processing method. This section will detail the different steps and techniques used in each.

J o u r n a l P r e -p r o o f

Viruses can partition to solids in wastewater, and this is more pronounced for enveloped than nonenveloped viruses 26 . Therefore, it was anticipated that some laboratories may be dissociating the viruses from the solids prior to treatment to improve their detection of SARS-CoV-2 or removing the solids for separate processing. A majority of 36 respondents to question 19 indicated they do not dissociate the viruses from solids in suspension (26; 72.2%). Of those performing virus dissociation, methods used included filtration, elution, sonication, and centrifugation ( Figure 3 ). Of the 35 responses to question 20 about preliminary solids removal, a majority of those indicated that they do conduct solids removal (25; 71.4%) either via centrifugation (14; 40.0%) or filtration (17; 48.6%), with six respondents saving solids for analysis. Analysis of these separated solids was conducted primarily by direct nucleic acid extraction (8) and/or by acid adsorption/elution (2), amino acid buffer extraction (1), and Vertrel TM purification (1).

Information was sought about the types of methods used for concentration of SARS-CoV-2 as many have been used over the years for WBE of other viral pathogens 6, [27] [28] [29] . Some methods may be more appropriate for certain use cases than others (depending on sample type, sample characteristics, anticipated viral load, cost, virus recovery, etc.) and at the time this survey was conducted method comparisons for SARS-CoV-2 concentration were not available. A majority (86.5%) of the 37 respondents answering question 21 conduct primary concentration while only 13.5% do not. Polyethylene glycol (PEG) precipitation is the most frequently used primary concentration method, followed closely by membrane filtration (Figure 4 ). There were 5 respondents who conduct primary concentration, but not secondary concentration. Of these, the primary concentration methods used included membrane filtration (1), centrifugal ultrafiltration with centricons (3), PEG precipitation (2), and skim milk flocculation (1).

The most frequently processed sample volume was 100 mL or less, yielding a final volume after concentration of 10 mL or less for most respondents ( Figure 5 ).

Of the 16 responses to question 22 about recovery efficiency, the most frequent response indicated that the recovery efficiency is unknown (7; 43.8%). Other responses stated that the recovery was ≥20% (1; 6.3%), ≥40% (2; 12.5%), ≥90% (4; 25.0%) or varied (2; 12.5%).

Approximately half of the 32 respondents to question 23 conduct secondary concentration (46.9%) and half do not (53.1%). The most frequently used secondary concentration method was centrifugal ultrafiltration, followed by PEG precipitation and skim milk flocculation ( Figure 6 ). Similar to the responses for primary concentration, a majority of the 7 responses to question 24 about recovery efficiency indicated that the recovery efficiency is unknown (4; 57.1%). Other responses stated that the recovery was <50% (2; 28.6%) or ≥70% (1; 14.3%).

There were 29 responses to question 25 about the use of purification, indicating most respondents do not conduct purification of the samples (26; 89.7%). Of the three respondents who do, two use Vertrel TM and one uses Vertrel TM and Sephadex TM . 

Choosing an appropriate detection method is crucial to obtaining usable results for the desired research or policy question, though some methods have limitations for use. For example, tissue culture will supply information on viable SARS-CoV-2, but is limited to laboratories with appropriate resources and containment facilities. Molecular detection is commonly used, but low levels of SARS-CoV-2 in samples can make quantification challenging. Understanding the techniques utilized can help future researchers identify those appropriate for their use case. A majority of the 31 respondents obtained quantitative detection results using RT-qPCR, ddRT-PCR, or tissue culture (28; 90.3%) rather than presence/absence results using RT-qPCR (7; 22.6%) (question 32). Of the detection methods asked about, molecular methods were the most frequently used (24/30; 80%; question 34) followed by minION or Sanger sequencing (4/21; 19%; question 36) and tissue culture using plaque assay, TCID50, or MPN (most 

The effective volume assayed is important to consider when selecting a sample processing method.

Larger effective volumes assayed can lead to increased likelihood of detection of a low concentration target but can also concentrate more inhibitors which can affect detection if using RT-qPCR. Seventeen (17) respondents answered question 39 about the effective volume assayed, with seven (41.2%) yielding an unknown answer. Of these answers, the effective volume assayed was 5 mL or less (11.8%: 2), 50 mL or less (23.5%; 4), 100 mL or less (5.9%; 1), 500 mL or less (11.8%; 2), and 1000 mL or less (5.9%; 1).

However, as calculations were not provided to determine these values, these should be used cautiously.

Techniques used to understand sampling and method functionality and consistency include field and transport blanks, recovery controls, initial precision recovery controls, ongoing precision recovery concentrations and with greater inhibition present. Since the survey was conducted in mid-April 2020 many laboratories have continued to develop and implement WBE approaches for SARS-CoV-2. This data helped inform the WRF International Summit in late April 2020, which enabled global experts to determine best practices and rapidly guide the design of a methods comparison study. In June 2020, 92 laboratories indicated that they had methods developed and were willing to take part in a comparative evaluation of methods (WRF project 5089) 22 . As further research is conducted, it is vital to consider development or optimization of methods applicable for use in lower-and middle-income countries (LMIC), which are more resource limited. Finally, as SARS-CoV-2 WBE studies have continued and methods improved over the past eight months since this survey was conducted, a follow up survey will be developed to determine optimized methods and focus on effective volumes processed, cost, and method effectiveness and applicability for LMICs. Comparative evaluation of methods will enable rapid identification of the most reliable and robust methodological approaches and increase the credibility of WBE as a supplemental tool to support public health and policy decision making responses to COVID-

Appendix A. WRF Survey of Microbiological Methods for Wastewater Surveillance 

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Evidence for Gastrointestinal Infection of SARS-CoV-2

Wastewater-Based Epidemiology: Global Collaborative to Maximize Contributions in the Fight Against COVID-19

Guidelines on environmental surveillance for detection of polioviruses

Environmental Surveillance for Polioviruses in the Global Polio Eradication Initiative

The Potential of Wastewater Testing for Public Health and Safety

International Water Research Summit on Environmental Surveillance of COVID-19 Indicators in Sewersheds

Estimating mortality from COVID-19: Scientific brief

SARS-CoV-2 RNA in wastewater anticipated COVID-19 occurrence in a low prevalence area

How sewage could reveal true scale of coronavirus outbreak

SARS-CoV-2 in wastewater: potential health risk, but also data source

Presence of SARS-Coronavirus-2 in sewage

Temporal detection and phylogenetic assessment of SARS-CoV-2 in municipal wastewater

SARS-CoV-2 RNA concentrations in primary municipal sewage sludge as a leading indicator of COVID-19 outbreak dynamics

Metropolitan Wastewater Analysis for COVID-19 Epidemiological Surveillance

FIRST DETECTION OF SARS-COV-2 IN UNTREATED WASTEWATERS

SARS-CoV-2 titers in wastewater are higher than expected from clinically confirmed cases

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

Detection of SARS-CoV-2 RNA in commercial passenger aircraft and cruise ship wastewater: a surveillance tool for assessing the presence of COVID-19 infected travellers

Reproducibility and sensitivity of 36 methods to quantify the SARS-CoV-2 genetic signal in raw wastewater: findings from an interlaboratory methods evaluation in the U

A comparison of SARS-CoV-2 wastewater concentration methods for environmental surveillance

Comparison of virus concentration methods for the RT-qPCR-based recovery of murine hepatitis virus, a surrogate for SARS-CoV-2 from untreated wastewater

CDCMMWR. COVID-19 Stats: COVID-19 Incidence, by Urban-Rural Classification -United States

Partitioning, and Recovery of Enveloped Viruses in Untreated Municipal Wastewater

Improvement of the Bag-Mediated Filtration System for Sampling Wastewater and Wastewater-Impacted Waters

New methods for the concentration of viruses from urban sewage using quantitative PCR

Comparison of Concentration Methods for Quantitative Detection of Sewage-Associated Viral Markers in Environmental Waters

The authors would like to thank the many respondents to this survey. 

The authors declare no competing interests.

Supplementary Information 1. Blank survey distributed.J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f