SARS-CoV-2 known and unknowns, implications for the water sector and wastewater-based epidemiology to support national responses worldwide: early review of global experiences with the COVID-19 pandemic

Wastewater surveillance of pathogens may be a useful tool to help determine whether clinical surveillance of disease is effective or inadequate due to under-reporting and under-detection. In addition, tracking of pathogen concentrations over time could potentially provide a measure of the effectiveness of public health control measures and the impact of the gradual relaxation of these controls. Analysis of wastewater using quantitative molecular methods offers a real-time measure of infections in the community, and thus is expected to provide a more sensitive and rapid indication of changes in infection rates before such effects become detectable by clinical health surveillance. Models may help to back-calculate wastewater prevalence to population prevalence or to correct pathogen counts for wastewater catchment-specific and temporal effects. They may also help to design the wastewater sampling strategy. This article provides a brief summary of the history of pathogen wastewater surveillance to help set the context for the SARS-CoV-2 wastewater-based epidemiology (WBE) programmes currently being undertaken globally.


GRAPHICAL ABSTRACT INTRODUCTION
Coronaviruses are a large and diverse family of viruses. The name 'corona' comes from their round appearance and the spikes on their surface that can be likened to a solar corona (Figure 1(a)). Coronaviruses are enveloped, which means that there is a lipid membrane envelope around the surface of the virus, while 'naked' viruses do not have this.
The lipid envelope makes coronaviruses more fragile than other viruses (Walls et al. ) and is hence relevant to understanding their resistance to disinfection, as well as their environmental persistence and transmission. The lipidic structure holds the membrane (M), envelope (E) and spike (S) proteins together, with the spike protein protruding around the envelope (Figure 1(a)). Since the spike protein is responsible for the connection with the host cells in humans, the virus loses its infectivity if the lipid envelope is destroyed ( Figure 1(b)) (Walls et al. ; Wu et al. a, b, c).
Another characteristic relevant to their sensitivity to UV disinfection is that the genome is made up of single-stranded RNA (Figure 1(a)). When looking for the virus in wastewater, scientists look for the genetic information that codes for the key proteins in its structure. Eurosurveillance and Centers for Disease Control and Prevention have provided references listing commonly used primers for the detection of SARS-CoV-2 virus. The Eurosurveillance E primers target regions and N2 primers detect fragments of RNA that code for the nucleocapsid (N) protein (Figure 1(a)).
The term HCoV is used to represent human coronaviruses. As most human coronavirus infections cause mild symptoms, they may even go unnoticed. Since the beginning of the 21st century, three coronaviruses have crossed the species barrier to cause deadly pneumonia in humans (Drosten et al. ; Zaki et al. ). These are: • Severe Acute Respiratory Syndrome (SARS-nCoV); • Middle-East Respiratory Syndrome (MERS); and now • Severe Acute Respiratory Syndrome 2 (SARS-CoV-2).
Six types of human coronavirus were identified before 2019, the seventh (SARS-CoV-2) was revealed after testing of fluid from a patient's lungs on 3 January 2020, following reports of several patients presenting with a strange pneumonia in November and December 2019 in Wuhan Province, China. The first publications about this virus referred to it as the 'novel coronavirus', and the name 2019-nCoV was used to denote it. Since more has become known about the virus, it has been designated SARS-CoV-2 and is associated with the current pandemic of atypical pneumonia (the disease is designated as COVID-19).
SARS-CoV-2 is transmitted from person-to-person via the respiratory system through sneezing, coughing and secretions, and by contact with contaminated surfaces (Huang et al. ; Zhu et al. ).
Coronaviruses belong to the family of coronaviridae, and the severe acute respiratory syndrome-related (SARS) coronavirus species includes the SARS-CoV-2 strain. This coronavirus is the newest of the family of coronaviruses associated with human infections that are grouped into the beta-CoV genus, with over 70% genetic similarity to SARS-  (Matrajt et al. ). Environmental surveillance has also been used and recommended for other infections, such as typhoid (WHO ), as well as for antimicrobial resistance (Hendriksen et al. ), with modelling techniques used to assist both the design and interpretation of those efforts (Wang et al. a, b). WBE is also commonly used in the surveillance of licit and illicit drugs and various chemical contaminants which may impact human health (Choi et al. ).
Evidence of pathogen concentrations in wastewater has been published and is now part of long-term routine monitoring programmes carried out by water utilities, and which utilise accredited laboratories for these analyses. In terms of viruses that are significant for the water sector, it is well-established that viruses are commonly found in wastewater, and hence routine testing for viruses often occurs. There are two main groups of viruses commonly found in wastewater. Firstly, viruses that are more resistant to natural and engineered inactivation processes Although there is limited data on the survival of SARS-CoV-2 in water because they behave similarly in aerosols, similar behaviour is likely for SARS-nCoV and SARS-CoV-2 in water and wastewater. SARS-nCoV was predicted to be very stable at 4 C in filtered tap water. SARS-nCoV was found to remain live in stools for 6 days at room temperature, and fragments of SARS-nCoV continued to be detected in wastewater for up to 3 days, making it less stable in wastewater than polio (

RESEARCH AND INVESTIGATION PRIORITIES
Two priorities for investigation and research have been identified by the water sector. Firstly, wastewater monitoring can provide a simple means to determine if SARS-CoV-2 is present and which subtypes predominate. Secondly, understanding the resistance of SARS-CoV-2 to water and wastewater treatment processes (noting that SARS-CoV-1 was shown to be sensitive to both chlorination and UV disinfection, and hence, SARS-CoV-2 is predicted to be readily inactivated and otherwise removed through the conventional treatment of water and wastewater, and also by the treatment processes used for the production of high-grade recycled water (Water Research Australia a)).  (Wang et al. a, b). Models are now being proposed not only to design a particular sampling strategy but also to allow strategies to be adapted to maximise the information gathered during the surveillance effort. Initial knowledge and practical constraints may mean that it is not possible to implement the most optimal sampling design, and changing disease patterns could require the sampling design to be adapted over time (Wang et al. a, b).
Note that WBE models have been used for at least the past 20 years, for example, a simulation model to evaluate poliovirus environmental surveillance efficiency was published near the turn of the century (Ranta et al. ).
These models included transmission models (e.g. duration of virus shedding) and impacts of environmental factors (e.g. sewer system fate of the poliovirus) on the surveillance results and, finally, effects of sampling and laboratory analysis. Using such models, the detection probability for small outbreaks can be maximised.

IMPLEMENTATION OF AUSTRALIAN WASTEWATER SURVEILLANCE PROGRAMMES FOR SARS-COV-2
Representatives from the Water Research Australia ColoSSoS project are members of several of the working groups of the global research effort (Water Research Foundation ). This ensures that Australia's national approach to wastewater surveillance can be aligned to international best practice guidelines as they are developed.
The key activities of the ColoSSoS project are designed to support the health agencies' response to the current pandemic by providing reliable and robust data on the presence of SARS-CoV-2 in wastewater catchments and by sharing knowledge among the global community.
To respond quickly, Australian water utilities adopted a strongly pragmatic approach upon making the decision to undertake a SARS-CoV-2 wastewater surveillance programme. The implementation of such surveillance in the State of Victoria is illustrative of this pragmatism: sampling commenced on 1 April 2020 at the two large wastewater treatment plants that collectively treat most of Melbourne's wastewater. Victoria had just entered stage three restrictions ('lockdown') and utility and sampling staff movements were tightly controlled because of the need to ensure the ongoing provision of critical water and wastewater services.
Observing the decline in the growth of case numbers in the northern hemisphere following similar lockdown controls (so-called 'flattening the curve'), it was recognised that the peak number of first-wave COVID-19 infections could be reached in Victoria in a matter of weeks.
In the context of this rapidly changing and highly constrained environment, the objectives for Victoria were to implement a sampling programme before peak case numbers of COVID-19 were reached and to include locations within putatively COVID-19 free wastewater catchments, and sites proximal to large metropolitan hospitals from which the wastewater effluent was expected to be more likely to contain the SARS-CoV-2 virus. The sampling of locations predicted to be relatively enriched with SARS-CoV-2 was given a high priority due to the need to validate methods using both laboratory-generated and actual field samples.
Consequently, Victoria's initial SARS-CoV-2 sampling programme was designed to: • Be rapidly implemented at well-established wastewater treatment plant influent and sewerage network sampling locations through grab sampling and auto-sampling without deviating significantly from existing sampling protocols. For the most part, sites were chosen to be within 200 km of Melbourne so that sample bottles could be transported to a centralised storage location (À20 C facility), resulting in a coverage of approximately 71% of Victoria's 6.6 million population.
• Place minimal additional burden upon utilities.
Optimisation of the sampling programme will consider the benefits of various stratified sampling designs involving increased sampling frequency and use of inline, flow-