key: cord-334299-0zn1z7rc
authors: Ahmed, Warish; Bivins, Aaron; Bertsch, Paul M.; Bibby, Kyle; Choi, Phil M.; Farkas, Kata; Gyawali, Pradip; Hamilton, Kerry A.; Haramoto, Eiji; Kitajima, Masaaki; Simpson, Stuart L.; Tandukar, Sarmila; Thomas, Kevin; Mueller, Jochen F.
title: Surveillance of SARS-CoV-2 RNA in wastewater: Methods optimisation and quality control are crucial for generating reliable public health information
date: 2020-09-30
journal: Curr Opin Environ Sci Health
DOI: 10.1016/j.coesh.2020.09.003
sha: 
doc_id: 334299
cord_uid: 0zn1z7rc

Monitoring for SARS-CoV-2 RNA in wastewater through the process of wastewater-based epidemiology (WBE) provides an additional surveillance tool, contributing to community-based screening and prevention efforts as these measurements have preceded disease cases in some instances. Numerous detections of SARS-CoV-2 RNA have been reported globally using various methods, demonstrating the technical feasibility of routine monitoring. However, in order to reliably interpret data produced from these efforts for informing public health interventions, additional quality control information and standardization in sampling design, sample processing, and data interpretation and reporting is needed. This review summarizes published studies of SARS-CoV-2 RNA detection in wastewater as well as available information regarding concentration, extraction, and detection methods. The review highlights areas for potential standardization including considerations related to sampling timing and frequency relative to peak fecal loading times; inclusion of appropriate information on sample volume collected; sample collection points; transport and storage conditions; sample concentration and processing; RNA extraction process and performance; effective volumes; PCR inhibition; process controls throughout sample collection and processing; PCR standard curve performance; and recovery efficiency testing. Researchers are recommended to follow the Minimum Information for Publication of Quantitative Real-Time PCR (MIQE) guidelines. Adhering to these recommendations will enable robust interpretation of wastewater monitoring results and improved inferences regarding the relationship between monitoring results and disease cases.

The current global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARSmillion cases of COVID-19 and >950,000 deaths worldwide as of 18 th 

Of the 18 studies, nine studies used either a whole (concentration to RT-qPCR) process control or 126 a molecular (RNA extraction to RT-qPCR) process control. Only eight of the studies tested 127 wastewater samples for the presence of PCR inhibitors (that impede amplification) using process 128 J o u r n a l P r e -p r o o f information in the published literature for WBE of SARS-CoV-2. This lack of reporting on quality 145 control has the potential to limit the interpretation and usefulness of the produced data for advancing 146 the WBE field, and ultimately implementing public health interventions. A list of variables that are 147 likely to impact the detection and quantification of SARS-CoV-2 RNA in wastewater and therefore the 148 sensitivity and accuracy of WBE for public health surveillance are shown in Table 2 . Scaling SARS-CoV-2 wastewater surveillance to deliver national programs will also 154 likely require that testing be performed not only by research laboratories but also by commercial 155 laboratories. Such a rapid expansion in testing capacity makes robust and reproducible methods and 156 quality control vital to produce actionable public health information. In view of this need, we recommend methodological and quality assurance approaches for SARS-CoV-2 RNA detection in 158 wastewater using molecular methods.

Sampling design is a pivotal factor for detecting SARS-CoV-2 RNA in wastewater. The concentration 162 of SARS-CoV-2 RNA in influent wastewater is expected to vary diurnally, based on defecation 163 frequency and timing, as well as the sampling technique and frequency. Defecation in the general 164 population is most frequent in the early morning compared to other times [24] . Therefore, wastewater 165 collected during periods of peak fecal loading may be more enriched in SARS-CoV-2 RNA than 166 wastewater generated at other times in the day. In situations where an autosampler is not available, 167 these periods of increased fecal loading should be targeted for grab sampling, as one or more grab 168 samples taken during these periods would provide a higher probability of SARS-CoV-2 RNA detection 169 and quantification owing to higher RNA concentrations. We recommend that peak fecal loading times 170 be identified prior to a sampling campaign dependent on grab samples. While the exact peak fecal 171 loading periods will vary between WWTPs due to differences in sewer infrastructure, total influent flow 172 may be a useful proxy for anthropogenic activity and fecal shedding in the morning. understood and should be avoided until more data are available. We acknowledge that pasteurization 214 is often undertaken to minimize the risk associated with handling wastewater. However, when the recommended as it may produce false negative results. We also encourage researchers to provide critical information on sample metadata such as biochemical oxygen demand (BOD), chemical 218 oxygen demand (COD), total suspended solid (TSS), pH, etc, storage temperature, storage time prior 219 to sample processing, whether the samples were frozen prior to virus concentration, as well as any 220 pre-treatment before the concentration step. should be taken to minimize overall inhibition. To achieve this, we recommend that each wastewater 256 sample should be seeded with a surrogate virus as a whole-process control to obtain information on 257 the surrogate virus recovery and RNA recovery and RT-PCR inhibition for the entire process starting 258 from sample concentration to RT-PCR detection [31] . If an appropriate process control is not 259 available, molecular process controls can also be included to obtain information on the RNA 260 extraction efficiency, and RT-PCR inhibition should, at a minimum, be evaluated (e.g., through target 261 dilution) and reported. Based on the process control data, samples may need to be reanalyzed (e.g.,

after dilution to reduce PCR inhibitors to suitable levels) or switch to an alternative method for 263 increased sensitivity [31].

For a reliable process control, it is appropriate to select a virus which is morphologically and 265 genetically similar to the target virus and is expected not to be present in the tested water. For 266 example, for SARS-CoV-2, low-pathogenic animal CoVs such as MHV, BCoV, FIPV or enveloped 267 bacteriophages, such as Phi6 phage, represent ideal controls. We acknowledge that obtaining a 268 suitable process control may be difficult for many laboratories, especially during a pandemic.

Nonetheless, the selection of already established process controls for enteric viruses, such as single- 

In order to obtain high quality RNA of SARS-CoV-2 for RT-qPCR analysis, several factors need to be 279 considered, including the RNA extraction procedures, the concentration, purity, and integrity of the 

While wastewater surveillance for SARS-CoV-2 has the potential to act as an early warning system, 329 the merit for WBE will be influenced by site selection and can only be realised with rapid turnround.

The value of results from wastewater surveillance decreases with increasing turnaround time, 331 particularly in cases where other methods do not provide rapid, objective information. To maximise 332 this value, sampling programs should aim to minimise the time take between the stages of approvals, 333 wastewater sampling, analysis, reporting and consequent actioning based on the results. We 334 recommend that each program develop its own unique operating protocols for each of these four 335 aspects in order to maximise the value of surveillance efforts. Sites may be selected for many 336 reasons based on the status of the pandemic in a particular region. The fast commencing of sampling will require an efficient approval process and close collaboration with those responsible for sewer 338 networks, such as city councils, water utility companies and service provider companies. In the case 339 where WBE is being applied to small populations, as may be the case with age care facilities, prisons, 340 and university campus accommodation, ethics approvals may be needed. Rapid response coupled 341 with rapid turnround clearly provides the best chance of capturing early detects. J o u r n a l P r e -p r o o f Declaration of interests ☐ X The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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