key: cord-0730571-v0z1vuw3 authors: Ahmed, Warish; Simpson, Stuart L.; Bertsch, Paul M.; Bibby, Kyle; Bivins, Aaron; Blackall, Linda L.; Bofill-Mas, Sílvia; Bosch, Albert; Brandão, João; Choi, Phil M.; Ciesielski, Mark; Donner, Erica; D'Souza, Nishita; Farnleitner, Andreas H.; Gerrity, Daniel; Gonzalez, Raul; Griffith, John F.; Gyawali, Pradip; Haas, Charles N.; Hamilton, Kerry A.; Hapuarachchi, Hapuarachchige Chanditha; Harwood, Valerie J.; Haque, Rehnuma; Jackson, Greg; Khan, Stuart J.; Khan, Wesaal; Kitajima, Masaaki; Korajkic, Asja; La Rosa, Giuseppina; Layton, Blythe A.; Lipp, Erin; McLellan, Sandra; McMinn, Brian; Medema, Gertjan; Metcalfe, Suzanne; Meijer, Wim G.; Mueller, Jochen F.; Murphy, Heather; Naughton, Coleen C.; Noble, Rachel T.; Payyappat, Sudhi; Petterson, Susan; Pitkänen, Tarja; Rajal, Veronica B.; Reyneke, Brandon; Roman, Fernando A.; Rose, Joan B.; Rusiñol, Marta; Sadowsky, Michael J.; Sala-Comorera, Laura; Setoh, Yin Xiang; Sherchan, Samendra; Sirikanchana, Kwanrawee; Smith, Wendy; Steele, Joshua; Subburg, Rosalie; Symonds, Erin M.; Thai, Phong; Thomas, Kevin V.; Tynan, Josh; Toze, Simon; Thompson, Janelle; Whiteley, Andy S.; Wong, Judith Chui Ching; Sano, Daisuke; Wuertz, Stefan; Xagoraraki, Irene; Zhang, Qian; Zimmer-Faust, Amity G.; Shanks, Orin C. title: Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance date: 2021-08-25 journal: Sci Total Environ DOI: 10.1016/j.scitotenv.2021.149877 sha: 2d3713c17a7abdbdcbf81e0920469c11697bac24 doc_id: 730571 cord_uid: v0z1vuw3 Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering additional community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of SARS-CoV-2 in wastewater can provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2, culminating in recommended strategies that can be implemented to identify and mitigate these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, amplification inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases. Coronavirus disease-2019 , a global pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in 202,293,191 of diagnosed cases and 4,286,447 deaths globally by August 8, 2021 (Dong et al., 2020) . SARS-CoV-2 is an enveloped respiratory virus with an RNA genome. Typical symptoms of COVID-19 may include any or a combination of the following symptoms: fever or chills, cough, shortness of breath or difficulty breathing, fatigue, muscle or body aches, headache, loss of smell/taste, chronic rhinorrhea, nausea or vomiting, and diarrhea in infected individuals (CDC, 2021) . Besides acute impacts, the virus can also cause debilitating late sequelae (CDC, 2020) . COVID-19 is commonly detected in symptomatic individuals by collecting nasopharyngeal or throat swabs (Wölfel et al., 2020) and analyzing for SARS-CoV-2 RNA using reverse transcription-polymerase chain reaction (RT-PCR); however, antibody-and/or antigen-based tests are also used as a clinical surveillance tool (Ward et al., 2020) . Notably, not all SARS-CoV-2-infected individuals develop the typical symptoms described above (Nishiura et al., 2020) . Asymptomatic individuals may account for approximately 40-50% of infections (Oran and Topol, 2020) . These individuals can transmit the virus to others in the community for >14 days (Gandhi et al., 2020; Oran and Topol, 2020) . Symptomatic and asymptomatic COVID-19 patients shed SARS-CoV-2 in their feces, saliva, nasal secretions, and urine (Cevik et al., 2020) . A study found that analysis of fecal samples for SARS-CoV-2 can be more sensitive to infection than using samples from the respiratory tract, as feces carried a greater viral load, and positive results were consistently obtained over the course of the disease (Yuan et al., 2021) . Intact or degraded SARS-CoV-2 viruses and RNA fragments reach sewer networks through toilets, showers, wash basins and sinks and can subsequently be detected in wastewater, both at the sub-catchment scale and in the influent to wastewater treatment plants (WWTPs) . Recent studies have highlighted the potential of SARS-CoV-2 RNA surveillance in untreated wastewater to provide early detection or act a warning system for COVID-19 circulation in a community (Ahmed et al., 2020a; Ahmed et al., 2020b; Kumar et al., 2020; La Rosa et al., 2020; Medema et al., 2020; Miyani et al., 2020; Randazzo et al., 2020; Rimoldi et al., 2020; Wu et al., 2020; Chavarria-Miro et al., 2021; Gerrity et al., 2021) . A caveat common to all methods based solely on the detection of J o u r n a l P r e -p r o o f Journal Pre-proof genetic material is that wastewater surveillance measures the presence and quantity of SARS-CoV-2 RNA, not viable or infectious viruses from the individual building level to large metropolitan areas (i.e., WWTP influents). This surveillance approach is currently used worldwide as a complementary tool for monitoring SARS-CoV-2 RNA in wastewater . Wastewater surveillance programs are not novel or restricted solely to detection of COVID-19; wastewater surveillance for poliovirus and hepatitis A virus are past and current practices (Asghar et al., 2014; La Rosa et al., 2014; Alleman et al., 2021) . SARS-CoV-2 wastewater surveillance is currently practiced in at least 55 countries ( Supplementary Fig. 1 ). Over 230 organizations have announced that they are analyzing wastewater samples for SARS-CoV-2 RNA, with samples being sourced from > 1,370 sites (including WWTPs, sewer catchments, and surface waters) (Naughton et al. 2021 ; https://arcg.is/1aummW). Potential benefits of wastewater surveillance include detecting pre-and asymptomatic cases and the possibility of screening large populations with a single or few samples, independent of clinical testing availability and willingness . With strategic facility-specific or sub-catchment sampling, wastewater surveillance can also identify COVID-19 transmission clusters within large geographic regions, and this information can facilitate focused and prompt action by local health authorities . Actions arising from positive results from wastewater surveillance might include public announcements to encourage individual testing, increasing the availability of testing, establishing fever clinics in specific locations, implementing stay-at-home directives, or mandating other mitigation measures such as face masks, social distancing, and capacity limits to reduce transmission (Randazzo et al., 2020) . An important aspect of wastewater surveillance is the ability gather data rapidly and cost-effectively from a large population of thousands of individuals, with enough potential sensitivity to detect a few infected individuals contributing SARS-CoV-2 RNA to the local wastewater system (Michael-Kordatou et al., holes, pumping/lift stations, or facility influent) or, for near-source surveillance, (i.e., outlets of buildings, followed by virus concentration, extraction of RNA, use of RT-PCR for SARS-CoV-2 RNA detection and quantification (RT-qPCR; based on a standard curve or RT-dPCR; digital PCR without the need for a standard curve), and data analysis and interpretation. Many factors can affect the overall method performance and the reliability of the results (Ahmed et al., 2020d; Bustin et al., 2009; Medema et al., 2020b; Pecson et al., 2021 ). An optimized RT-PCR assay can theoretically detect a single SARS-CoV-2 RNA fragment (Bustin et al., 2009) . Such a high sensitivity is particularly useful for SARS-CoV-2 surveillance in wastewater where the RNA target concentration can be extremely low (~20 to 2,000 GC/L) (Ahmed et al., 2020a; Haramoto et al., 2020) . However, increased analytical sensitivity coupled with poor RT-PCR assay specificity can increase the likelihood of false-positive errors. In this instance, a false-positive occurs when an RT-PCR result is positive for SARS-CoV-2 RNA, whereas it is absent in the wastewater sample. While false-positive errors can be monitored, minimized, and eliminated through strict adherence to best laboratory practices with appropriate controls, false-negative outcomes are more difficult to identify and manage. A false-negative error occurs when RT-PCR fails to J o u r n a l P r e -p r o o f Journal Pre-proof detect SARS-CoV-2, but the target RNA is actually present in the wastewater sample. Falsenegative errors may arise from various factors inherent to a particular wastewater sample, including how it is collected, transported, and stored (i.e., freezing), the low abundance of the RNA target in wastewater, or the degradation of the RNA target in the sewer collection system. Other factors are related to sample processing and analysis, such as poor virus recovery and/or low effective sample volume (ESV) assayed, low RNA extraction efficiency, amplification inhibition in the RT-PCR assay, and poor assay sensitivity (Ahmed et al., 2020d; Medema et al., 2020b; Michael-Kordatou et al., 2020) . In this study, we discuss: (i) the implications of SARS-CoV-2 false-positive and falsenegative errors for public health responses, (ii) factors that may cause false-positive and false-negative errors of SARS-CoV-2 RNA in untreated wastewater across the entire wastewater surveillance process, and (iii) strategies that can be used to minimize the likelihood and subsequent impacts of false-positive and false-negative errors on stakeholder confidence in SARS-CoV-2 wastewater surveillance. In this study, we provide a basic scientific framework that can aid in setting and developing appropriate quality control measures around sampling and laboratory workflows regarding wastewater surveillance for SARS-CoV-2 and other pathogens. Typically, for an analytical test such as RT-PCR, false-negative and false-positive errors are interpreted only regarding the presence or absence of the relevant analyte in the sample, as mentioned above. However, during wastewater surveillance, such as for SARS-CoV-2, the RT-PCR results are interpreted as relevant to two outcomes: (i) the presence or absence of SARS-CoV-2 RNA in a wastewater sample and, subsequently, (ii) the presence or absence of SARS-CoV-2 infections in the community. Hence, two layers of error that must be considered. The first layer, which we termed RT-PCR false-positive or RT-PCR false-negative is pertinent to whether SARS-CoV-2 RNA is present or absent in a wastewater sample, and whether RT-PCR test results accurately reflect that status. The second layer, termed wastewater surveillance false-positive or false-negative, is pertinent to whether SARS-CoV-2 infection is J o u r n a l P r e -p r o o f Journal Pre-proof The wastewater collection process presents multiple opportunities to contaminate samples, which could result in false-positive errors. SARS-CoV-2 RNA can persist in untreated wastewater and tap water for approximately 10 days, at 37°C or approximately 30-60 days, at 4°C (Ahmed et al., 2020c) . In regions with a high prevalence of COVID-19, SARS-CoV-2 RNA has been found on various surfaces in public places (Gholipour et al., 2020) . Therefore, SARS-CoV-2 RNA can potentially accumulate on the surface of sampling equipment (e.g., tubes attached to autosamplers and, contaminated sampling bottles) and gear (e.g., telescopic sampler, pump, and multi-meters). Consequently, wastewater samples could become contaminated due to inadequate cleaning between equipment uses. A false-positive error can also occur when handling multiple samples at an individual collection location (near the influent or maintenance hole etc.) due to gloves and clothing being contaminated by accidental splashes or possibly aerosols. Improper storage during transport to the laboratory also could cause cross-contamination. Ice inadvertently contaminated by positive samples could potentially transfer SARS-CoV-2 RNA to another sample if the bottle lids were not properly sealed. Sampling guidance from government agencies such as USEPA, describe quality assurance and quality control protocols that are vital for assessing such contamination, including sample container cleanliness, equipment to be used, and sample negative controls (USEPA 2007). Sample-to-sample contamination may also occur in the laboratory during sample processing and storage (i.e., in a cold room or a refrigerator), sample concentration, RNA extraction from positive samples, or from positive control material (i.e., synthetic fragments, gammairradiated, or heat-inactivated SARS-CoV-2 reference materials), and/or RT-PCR amplification steps and amplicons generated from previous SARS-CoV-2 positive samples. Also, cross-contamination from an analyst's personal protective equipment to laboratory surfaces has been identified using RT-droplet digital PCR (RT-ddPCR) assays (Lv et al., 2020) . A shortage of skilled laboratory personnel due to the high demand for analysts has resulted in less experienced individuals working in laboratories, which could exacerbate J o u r n a l P r e -p r o o f cross-contamination issues and false-positive errors (Albano et al., 2020; Giri et al., 2020) . Such issues may affect clinical diagnostics and wastewater surveillance, especially in areas with a low incidence of COVID-19. Some laboratories are analyzing numerous wastewater samples (up to 100 or more per week) for routine surveillance, and the testing rates may increase. As interest grows in routine wastewater surveillance, public health units that utilize the data for management actions demand faster results output. Both factors (sample numbers and rapid output) place increased pressure on laboratory personnel, which may lead to increases in errors potentially resulting in increases in contamination and false-positive errors. False-positives can also originate from the RT-PCR reagents themselves or due to poor assay specificity. The notion that commercial PCR reagents can contain unwanted contamination is not new. There are numerous reports of E. coli fragments in DNA polymerase preparations (Koponen et al., 2002) and human mitochondrial DNA fragments in deoxyribonucleotide preparations (Wilson et al., 2016) . These contaminants are introduced during the manufacturing of reagents and, in some instances, can lead to false-positive errors. False-positive errors of SARS-CoV-2 assays in clinical specimens have been reported in Europe, where several laboratories have identified commercial primer and probe reagent batches contaminated with synthetic control materials (Mögling et al., 2020) . Improper selection of SARS-CoV-2 target genes or design of primers and probes that are not specific to the target may also lead to false-positive errors. Previous testing of seven different RT-qPCR primer and probe sets on clinical samples found that all the assays, including the US CDC N1, N2, and N3 assays, were "highly specific" for SARS-CoV-2 with no observed cross-reactivity (Nalla et al., 2021) . However, environmental sample matrices are much more complex and dynamic presenting unique challenges for RT-qPCR assays compared to clinical samples, and observations of specificity have not been consistent for wastewater testing. For example, Westhaus et al. (2020) noted that certain SARS-CoV-2 RT-PCR assays targeting the RdRP or M-gene, may also detect other endemic human coronaviruses, leading to false-positive errors for SARS-CoV-2. Gerrity et al. (2021) reported an instance of cross-J o u r n a l P r e -p r o o f reactivity between the E_Sarbeco assay and seeded bovine coronavirus. Pecson et al. (2021) reported significant cross-reactivity between a human coronavirus OC43 assay and seeded bovine coronavirus-two common SARS-CoV-2 surrogates used as process controls. Thus, it is essential that the specificity (i.e., amplification of only the target SARS-CoV-2 gene) of the selected SARS-CoV-2 target sequence and the RT-PCR oligonucleotides are adequately assessed to increase confidence in SARS-CoV-2 RNA detection and minimize the potential for false-positives errors. Confirmation of assay specificity is particularly relevant for assays targeting emerging SARS-CoV-2 genome variants of concern, such as the B.1.1.7 (Alpha), P.1 (Gamma), B.1.351 (Beta), and B.1.617.2 (Delta) variants. These variants of concern are characterized by spike protein substitutions that can increase transmissibility and disease severity and may interfere with diagnostic assay performance (https://www.cdc.gov/coronavirus/2019-ncov/variants/variant-info.html#Concern). For example, early in the emergence of the Alpha variant, a S-gene "dropout" during RT-qPCR testing with certain kits indicated the presence of this mutation and the potential presence of the B.1.1.7 lineage (https://www.thermofisher.com/blog/behindthebench/solutions-forsurveillance-of-the-s-gene-mutation-in-the-b117-501yv1-sars-cov-2-strain-lineage/). Since then, RT-qPCR assays specific to new variants have been developed by targeting mutations in various genomic regions, predominantly the spike protein gene. For example, an allelespecific RT-qPCR assay for the Alpha variant was developed with "low cross-reactivity" and successfully used to test wastewater samples from communities in the United States . Another study reported the development of sensitive and specific RT-qPCR assays for the detection and quantification of both the Alpha and Beta variants of concern in wastewater (Yaniv et al., 2021) . In the Netherlands, RT-droplet digital PCR (ddPCR) and an assay specific for mutation N501Y were used to detect the circulation of the Beta variant with proportions as low as 0.5% in a wastewater sample (Heijnen et al., 2021) . Given the mutation rates inherent to viral replication, variants of concern are likely to continue to emerge. For example, more recently a RT-qPCR assay was developed targeting Gamma (P.1) and Delta (B.1.617 variants (Yaniv et al., 2021) . Confirming the specificity of novel RT-qPCR assays for detecting and quantifying such variants in wastewater will be vital for effective future wastewater surveillance. J o u r n a l P r e -p r o o f SARS-CoV-2 false-negative errors in wastewater surveillance can result from inefficient RNA recovery in samples, PCR inhibition, or methods not optimized to detect trace quantities of target RNA. This section will review known and possible sources of false-negative errors for RT-PCR wastewater surveillance applications. Individuals infected with SARS-CoV-2 shed the virus and its genetic material via their sputum, nasopharyngeal secretions, saliva, urine, and feces (Cevik et al., 2020) . SARS-CoV-2 RNA concentration in the feces of infected individuals is highly variable and ranges from 10 2 to 10 9 GC/g of feces (Wölfel et al., 2020; Zhang et al., 2020) . The SARS-CoV-2 RNA detection rate in feces can range from 11 to 83% among COVID-19 positive adults (Wölfel et al., 2020) and up to 100% among children . Therefore, as the prevalence of COVID-19 decreases within a community, the number of individuals shedding the virus in their feces, urine, and nasal secretions and the subsequent amount of RNA shed into the sewer system will also decrease, thus reducing the amount of virus in the wastewater system. The low and sporadic occurrence of SARS-CoV-2 RNA in wastewater due to low COVID-19 cases in the community makes RNA more challenging to detect. Therefore, it is critical to take steps to minimize the likelihood of false-negative errors. The effects of RNA target dilution, in large part due to mixing with other wastewater flows, stormwater infiltration, diurnal variation in shedding, and hydraulic residence time in the sewer collection system, can also affect the probability of detecting SARS-CoV-2 RNA, particularly in low prevalence conditions (Ahmed et al., 2020d (Gonzalez et al., 2020; Gerrity et al., 2021; Wu et al., 2021) . Despite mixing and dilution in sewer systems, it is incorrect to consider sewage, as it arrives at a WWTP to be a well-mixed, evenly distributed liquor of everything discharged to the system during the day (Teerlink et al., 2012) . Instead, sewers are dynamic flow systems, and the composition of wastewater arriving at a WWTP changes continually, reflecting variations in flow rates and substances discharged into the wastewater system (Ort et al., 2010b) . Some wastewater systems can provide a degree of lateral flow mixing, such as those with storage capacity associated with pumping stations or flow equalization tanks. Turbulent flow regimes in sewer pipes can also induce lateral mixing, but this homogenization is often limited and may not be substantial relative to the overall volume of wastewater conveyed. For a substance that is constantly or frequently discharged into a sewer network in relatively constant amounts, any random wastewater sample could reasonably be used to estimate a daily load. However, for a substance that is sporadically discharged into sewers in varying amounts temporally, either continuous sampling or multiple samples collected at different times of the day will be required to accurately estimate the total daily load (Ort et al., 2010a) . Discontinuous surveillance using grab samples imparts a high probability of a falsenegative error occurring or even disproportionately high concentrations that do not accurately J o u r n a l P r e -p r o o f reflect conditions in the broader community. Under low-prevalence conditions, if a sample is not collected at the precise moment that the wastewater "slug" containing a target substance (i.e., SARS-CoV-2 RNA) passes through the sampling point, "non-detection" may occur (Ort et al., 2010b) . This concept is illustrated in Fig. 2 detectable dispersion volumes will vary depending on the parameters of different sewer systems (e.g., SARS-CoV-2 RNA persistence given sewer travel times and temperatures, and mixing). The implication is that there is a greater likelihood of a positive detection for a larger detectable dispersion volume. Since the dispersion volumes of multiple individuals may overlap, the probability P (x) of any small-volume grab sample containing the detectable RNA excreted from any number of multiple individuals can be considered using the binomial distribution P (x) ~ B (n, p), where x is the specified number of individuals for which excreted RNA can be detected in that sample, n = the number of individuals shedding SARS-CoV-2 RNA, and p = the probability that the sample contains a detectable concentration of a specific individual's excreted SARS-CoV-2 RNA (as given by the ratio above). Given the assumed parameters and model described above, the probability of a single wastewater sample being negative for SARS-CoV-2 RNA is given by the binomial distribution solution for P (x = 0), as shown in Fig. 3a . If the proportion of people shedding SARS-CoV-2 is <0.01%, the likelihood of a grab sample not containing the RNA at a detectable concentration is above 90% for all three assumed mixing scenarios. In this case, x=0, n<10 people per 100,000, and p depends on the average detectable dispersion volume of each person's excretions. In any of the circumstances considered in Fig h study of wastewater influent in Indiana, USA, only 50% of grab samples (collected every two hours) were positive for SARS-CoV-2 RNA when new COVID-19 cases averaged 16/day, but 100% of samples were positive when new cases averaged 65/day (Bivins et al., 2021a) . During the same study, an increasing number of COVID-19 cases in each community was also associated with decreasing variation in SARS-CoV-2 RNA concentration over 24 h of surveillance (Bivins et al., 2021a) . CoV-2 RNA is assumed to be a sporadically discharged substance-an assumption only applies where the proportion of concurrent excreters is very low. However, it may be relevant in any circumstance where wastewater surveillance is conducted for providing an "early warning" of the re-emergence of COVID-19, following near elimination of the virus. In such cases, it is important to recognise that failures to detect RNA in wastewater do not provide infallible evidence that RNA and COVID-19 infected people are not sporadically present in wastewater and the community, respectively. Importantly, the model presented here is purely conceptual and was developed to illustrate the considerations for achieving representative wastewater sample for COVID-19 surveillance. Also, the model input parameters and outcomes may vary greatly considering different types of wastewater collection systems and shedding rates. An additional consideration is that the actual behavior of SARS-CoV-2 in wastewater collection systems remains poorly characterized. Enveloped viruses have been observed to partition favorably to solids, which likely changes their transport behavior from the dissolved species frequently used to characterize fate and transport in hydraulic systems (Ye et al., 2016) . The adsorption of SARS-CoV-2 to surfaces such as suspended solids depends on both surface chemistry and solution chemistry, which further complicates the idealized conceptualization of fate and transport in wastewater . Additionally, SARS-CoV-2 and its RNA have been observed to exist in several forms in wastewater, including intact and ruptured viruses (Wurtzer et al., 2020) . Since release of the first preprint paper on detecting SARS-CoV-2 in wastewater (Medema et al., 2020a) , selecting the best sampling strategy has been a challenge for wastewater surveillance studies. As described above, poorly designed wastewater sampling strategies can easily contribute to false-negative errors (regardless of how good the laboratory workflow is) (Ahmed et al., 2020d) . Common sampling approaches include grab samples, which may be individual samples or composites of several grabs, or the use of an auto-sampler to collect a composite sample over a longer time, frequently 24 h (WHO, 2003; Miyani et al., 2020) or the use of passive samplers where an absorbent material such as swabs or medical gauze is J o u r n a l P r e -p r o o f placed in the wastewater and retrieved after hours to days, as a low-cost alternative to autosamplers (Schang et al., 2021) . In most scenarios, samples created from a composite of multiple sub-samples offer a more representative measure of viral concentration in wastewater (Matrajt et al., 2018) . Composite auto-samplers deployed at fixed sampling locations are becoming the most favored sampling approach in high-income countries (Hamouda et al., 2021) . Depending on the wastewater system, either time-weighted or flow-weighted composite sampling may be more effective for capturing concentration spikes of pharmaceuticals and personal care products (Ort et al., 2010b) . Only in systems where wastewater is homogenized and dilution is limited grab sampling may result in representative samples (e.g., sampling from wastewater systems of airplanes and cruise ships) (Ahmed et al. 2020f; Albastaki et al., 2021) . Sampling small wastewater collection networks (e.g., buildings or neighborhoods), where few toilet flushes occur for a day and large short-term fluctuations are expected, would require an increased sampling frequency or sampling to target the peak flow periods (e.g., during the morning hours) to capture these shedding events (Aymerich et al., 2017) . Passive samplers may be useful alternatives in such cases, as they have a demonstrated the ability to accumulate pathogens over time, often with greater sensitivity and efficiency than grab samples. Passive samplers are much less costly and can be much easier to deploy in drains or maintenance holes with low flow conditions than autosamplers and have been used to detection SARS-CoV-2 RNA in wastewater (Hayes et al., 2021; Schang et al., 2021) . However, quantification of SARS-CoV-2 RNA per volume of wastewater may be difficult to achieve using passive samplers. Current good laboratory practices recommend that the concentration of wastewater samples be performed in a Biological Safety Level 2 (BSL-2) or Biological Containment 2 (BC2) laboratory with unidirectional airflow and BC3/BSL-3 precautions (CDC, 2019). Pasteurization can be used to minimize biosafety risk and help ensure safe sample handling, especially in instances of sample manipulation that may generate aerosols (i.e., sample concentration) (Whitney et al., 2020; Wu et al., 2020) . However, temperature, in general, can significantly influence microbial decay (Espinosa et al., 2020; Korajkic et al., 2019; Muirhead et al., 2020) 4 ). Significant and consistent reductions in N1 and N2 concentrations were observed from the three WWTPs, with reductions from 1 to 3 log 10 when the samples were concentrated by membrane adsorption coupled with the bioMerieux magnetic bead extraction kit (Fig. 4) . Given the inconsistent observations on the effect of pasteurization on SARS-CoV-2 RNA copy numbers, definitive recommendations regarding the use of heat treatment cannot be made at this time. Considering the biosafety requirements of the analytical laboratory, such effects should be characterized locally, and an appropriate course of action should be taken. J o u r n a l P r e -p r o o f samples was censored below the detection limit; values that were detected but not quantifiable (DNQ) were replaced with half the limit of quantification for statistical analysis. Samples in which the target was not detected (ND) were assigned a value of 1.0. Data were obtained partially from Steele et al. (2021) . Storage of wastewater samples at freezing temperatures results in a freeze-thaw cycle for the virus particles and can lead to SARS-CoV-2 RNA degradation, thus influencing the possibility of false-negative errors (Steele et al., 2021) . For example, some researchers have reported ~90% loss of SARS-CoV-2 RNA signal following storage at -80°C for one week (Weidhaas et al., 2020) . However, longer storage at freezing temperatures does not seem to cause additional loss of the SARS-CoV-2 RNA signal (i.e., no appreciable decay within 58 days at low and ultralow temperatures) (Hokajärvi et al., 2021) . A recent study also reported no significant decay of the OC43 RNA signal (another betacoronavirus, and a recognized SARS-CoV-2 surrogate) at -80°C over three weeks following multiple freeze-thaw cycles in an J o u r n a l P r e -p r o o f elution solution (containing 0.01% sodium hexametaphosphate, 0.01% Tween 80 and 0.001% Antifoam Y-20) (McMinn et al., 2021) . Together, these observations suggest that while freeze-thaw is a potential contributor to SARS-CoV-2 RNA signal decay, the storage state and conditions are likely important and contribute to the magnitude of signal loss. Viral pathogens are typically diluted in wastewater, requiring the sampling of large volumes (e.g., > 1 L) followed by a concentration step to obtain detectable amounts of viral nucleic acid (Ahmed et al., 2015) . Therefore, efforts to concentrate SARS-CoV-2 RNA from wastewater samples can be a potential source of false-negative errors due to losses incurred However, with HFUF or Centricon ® alone, the ESV decreased to ~1 mL, and virus recovery increased to > 50%. Therefore, when attempting to increase method sensitivity, one must simultaneously balance ESV and virus recovery, while also considering the practical aspects (e.g., time and cost) of sample collection and processing. A method comparison study revealed a 7-log 10 range of recovery efficiency and ALOD values, demonstrating the broad range of outcomes possible with currently available protocols and highlighting the importance of characterizing performance before implementation (Pecson et al., 2021) . Although, the study did not consider whether one approach surpassed another, it highlighted the importance of method characterization, including potential trade-offs between the sample concentration factor and virus recovery. Nonetheless, extremely low ESVs and virus recoveries have a greater potential to yield falsenegatives or even artificially inflate-adjusted concentrations. Inefficient RNA extraction procedures can contribute to the lack of detection of SARS-CoV-2 false-negatives, and little is known about which protocols are best for wastewater surveillance applications. Due to the COVID-19 pandemic, an overwhelming demand for materials and J o u r n a l P r e -p r o o f reagents for SAR-CoV-2 testing has resulted in manufacturer shortages, potentially requiring laboratories to quickly modify method workflows to accommodate short-term supply issues (Fomsgaard and Rosenstierne, 2020) . In some cases, researchers have been forced to process samples using a range of alternative/uncommon RNA extraction kits or to use methods lacking an RNA extraction step, all with varying levels of success (Fomsgaard and Rosenstierne, 2020; Merindol et al., 2020; Torii et al., 2020; Wee et al., 2020) . Commercially available RNA extraction kits can vary in efficiency and consistency in isolating viral RNA and as well as efficacy in removing PCR inhibitors, even when sourced from the same supplier (Iker et al., 2013; Griffin et al., 2014; Zhang et al., 2018) . Pivoting to alternative methodologies combined with potential inconsistencies in RNA recovery, even from commercial kits, the RNA extraction step remains a potential risk for false-negative detections due to low efficiency. Furthermore, with indications of the emergence of SARS-CoV-2 variants around the world, confounded by delays in vaccine distribution, manufacturer shortages of these critical reagents will likely remain an issue for ongoing wastewater surveillance efforts. Municipal wastewater is a complex, often non-homogenous, mixture of substances beyond fecal waste, including pharmaceuticals, personal care products, stormwater, sediments, household detergents, industrial effluents, metals, and other substances. Some of these substances may completely or partially inhibit the RT-PCR amplification process, leading to a false-negative result. These substances are generally referred to as PCR inhibitors and include a heterogeneous and poorly defined group of chemical substances known to inhibit PCR-based methods, including multi-ringed polysaccharides (e.g., humic and fulvic acids), fats, proteins, metal ions (e.g., iron and aluminum), RNases, and others (Schrader et al. 2021 ). There are multiple mechanisms of amplification inhibition, including co-precipitation of inhibitors with nucleic acids, either degrading or sequestering target nucleic acids; binding of some inhibitors to nucleic acids or enzymes, inhibiting polymerase activity; or chelating metal ions (i.e., Mg 2+) necessary for optimal amplification performance. A full description of known mechanisms was reviewed by Schrader and colleagues ( The SARS-CoV-2 RNA genome offers many possible targets for RT-PCR assays, including the nucleocapsid (N), envelope protein ( Institute (NMI) SARS-CoV-2 reference materials (https://www.industry.gov.au/news/newaustralian-standard-helps-covid-19-hotspot-detection), indicating the differences in assay performance based on paired testing of wastewater dilutions with the CDC N1 and N2 assays. Finally, a study conducted in Germany reported increased sensitivity and specificity of IP2/IP4 (RdRP) and M gene targets compared to N gene and E gene assays (Westhaus et al., 2020) , but cautioned that these assays might detect other endemic human coronaviruses, potentially inflating reported performance metrics. Although no study to date has conducted a head-to-head comparison of all SARS-CoV-2 assays in wastewater samples, the limited information available clearly indicates that some assays may be more suitable than others. J o u r n a l P r e -p r o o f PCR performance characteristics, such as sensitivity or assay limit of detection or quantification (ALOD or ALOQ), calibration model parameters for RT-qPCR (amplification efficiency, slope, Y-intercept), and dynamic range play an important role in interpreting the assay robustness and reproducibility, thus minimizing the potential for ambiguous results and detection failure (Bustin et al., 2009; Johnson et al., 2014; Borchardt et al., 2021; Bivins et al., 2021b) . ALOD is the concentration of a target that can be detected with a 95% probability (at least 95% of samples containing that quantity of target will be positive) (Bustin et al., 2009) . ALOQ for RT-qPCR can be defined in several ways, but in general, it depends upon the standard deviation around the lower concentrations of the standard curve. A relatively common practice is to establish the ALOQ based on the coefficient of variation (CV) for measuring analytical standards near the limit of detection. Acceptable CV levels are generally 10 to 35% (Forootan et al., 2017; Haugland et al., 2016) . For example, an assay in which the 100 GC standard has a CV of 15% and the 10 GC standard has a CV of 75% would have a limit of quantification of 100 GC. PCR-based assays with low ALOD and a wide dynamic range are ideal for wastewater surveillance applications. As previously described, MLOD is the method sensitivity or a concentration of a target that can be detected consistently after incorporating loss through the entire process from sample concentration to extraction, while the method limit of quantification (MLOQ) is the concentration of a target that can be quantified with an acceptable level of precision when present in a sample. Therefore, these values are calculated from the analytical limit of detection (ALOD) or quantification (ALOQ). SARS-CoV-2 RNA can undergo strong selection pressures, high mutation rates or a combination of both, resulting in the formation of several prominent genetic variants since late 2020 . Changes in key SARS-CoV-2 loci could result in reduced RT-PCR performance, leading to false-negative results depending on the location of the mutation relative to primers and probe-target regions. The rate of genetic change varies across the SARS-CoV-2 genome, with structural domains (i.e., spike, membrane, envelope, and J o u r n a l P r e -p r o o f nucleocapsid proteins) generally undergoing stronger selection pressure (due to host immunity) resulting in the accumulation of more mutations compared to nonstructural genes (i.e., ORF1a and 1b) (https://virological.org). One pertinent example is the emergence of the B.1.1.7 lineage (https://virological.org). However, accumulation of mutations within the probe and/or primer target regions is the utmost concern for wastewater surveillance, as these could, over time, lead to decrease in the sensitivity of RT-PCR detection, and an increased rate of false-negative errors. For example, a recent report indicated that single nucleotide polymorphism (SNP) in the nucleoprotein (N) gene interfered with some SARS-CoV-2 RT-PCR assays in clinical testing but not others (Ziegler et al., 2020) . Another group reported the failure of the RT-PCR assay, targeting the E gene due to an SNP (Artesi et al., 2020) . GISAID (Elbe et al., 2017) RT-PCR (both RT-qPCR, -dPCR and -ddPCR) has become a mainstream detection method for SARS-CoV-2 RNA in wastewater (Barcelo, 2020) . However, like all technologies, RT-PCR is not without limitations; in particular, reliance on a standard curve for RT-qPCR quantification and issues with amplification inhibition can be problematic. In recent years, digital PCR (dPCR) has emerged as an attractive alternative for environmental applications, as it offers absolute quantification without the need for a standard curve and may be more sensitive and less prone to inhibition (Cao et al., 2015) . As applied to the detection of SARS- CoV-2, RT-dPCR outperformed RT-qPCR for clinical specimens, including nasopharyngeal samples (Falzone et al., 2020; Liu et al., 2020; Suo et al., 2020) and plasma from infected patients (Tedim et al., 2021) , as it was found to be more sensitive and accurate. of RT-dPCR technology to quantify SARS-CoV-2 from municipal wastewater but offered no direct comparison with RT-qPCR (Gonzalez et al., 2020) . Notably, the potential for inhibition was assessed by adding a hepatitis G RNA control, resulting in approximately 50% recovery of the seeded material, suggesting that RT-dPCR may also be affected by interference from PCR inhibitors in wastewater. Another study reported the potential inhibition of RT-dPCR compared to RT-qPCR when applied to the quantification of SARS-CoV-2 from wastewater sludge samples (D'Aoust et al., 2020) , but this inference was based on a higher frequency of target detection by the RT-qPCR, rather than the recovery of an exogenous control. A direct comparison between RT-qPCR and RT-dPCR applied to aircraft and cruise ship wastewater samples indicated minimal differences in SARS-CoV-2 detection frequencies, with no inhibition observed with either approach. The authors concluded that potential differences in the detection frequency of SARS-CoV-2 may be considerably influenced by individual RT-PCR assay design rather than detection technology (Ahmed et al., 2020f) . Another research group performed a comparative study using both RT-ddPCR (droplet digital PCR) and RT-qPCR molecular workflows for quantifying the US CDC N2 gene in influent wastewater from several WWTPs (Fig. 8) . A positive signal was detected by RT-ddPCR at an earlier date when SARS-CoV-2 levels were presumably lower compared to RT-qPCR analyses. The capability for earlier detection was reinforced by comparing the analytical sensitivity of the two methodologies. Analysis indicated that the ALOD for N2 using RT-ddPCR was 0.25 copies/reaction whereas the ALOD for RT-qPCR was much higher at a concentration of 60.22 copies/reaction. While RT-ddPCR may be more sensitive than RT-qPCR for SARS-CoV-2 detection in wastewater, additional studies are needed to confirm these initial observations. Park et al. (2020) suggested relying on RT-ddPCR for quantification of reference materials and diagnosis in patients but utilizing RT-qPCR to analyze wastewater samples. A potential limitation of dPCR is the subjective thresholding that distinguishes positive partitions from negative partitions and the much narrower dynamic range of the measurement system. Since the entire sample often cannot be analyzed, replicated subsamples may be used to detect or estimate the concentration of a target DNA/RNA present in a single sample. Technical replicates test the variability of the analysis method and involve taking multiple subsamples from the same sample and analyzing each subsample. In contrast, biological or matrix replicates test different independently obtained wastewater samples. In general, the analysis of the three biological replicates can provide robust results. However, even if three samples were obtained for RNA extraction in an environmental application, only a subsample (a portion) of the extracted material from each sample was used in any individual RT-PCR reaction. This sub-sampling practice can introduce errors in RNA detection (Taylor et al., 2019) . If a 1-µL subsample of RNA is taken from a total volume of 100 µL of RNA containing a total of 500 GC of SARS-CoV-2 RNA, the expected number of GC in 1 µL of homogenous RNA subsample will be 5. However, if the quantity of SARS-CoV-2 RNA is 50 GC in a volume J o u r n a l P r e -p r o o f of 100 µL RNA, the expected number of GC in the 1 µL subsample will be 0.5. Assuming a theoretical ALOD of 1 GC, the latter scenario has a 50% probability of a false-negative error, assuming a single RT-PCR reaction. Therefore, a sub-sampling error is expected to be more pronounced in wastewater samples with low target RNA concentrations. It has been reported that subsampling error contributes to more than 10% of the variance when the concentration of a target in a subsample is below 100 GC, and above 30% when the concentration is below 10 GC. Technical variability of RT-qPCR assay results is reported to increase when yielding Cq values > 30 due to stochastic amplification, measurement uncertainty, and subsampling error (Taylor et al., 2019) . For example, considering the detection rate of SARS-CoV-2 using the N1 assay in 523 wastewater samples in Queensland, Australia (Pers. Comm. Phil M. Choi; Queensland health), the Inclusion of six rather than three technical replicates resulted in a ~45% detection frequency increase, suggesting that sub-sampling error and low replicate numbers can contribute to false-negative errors. Data reporting practices merit special consideration for highly infectious and transmissible viruses (and other biological agents), such as SARS-CoV-2, which present serious threats to global public health. For example, in instances where it has been documented that RT-PCR detection is not a result of extraneous contamination (i.e., false-positive), reporting detections below an established ALOQ such as detectable but not quantifiable (DNQ) (Layton et al., 2013 ) may be acceptable. In this case, the results below the ALOQ/MLOQ are classified as positive, thereby increasing the assay sensitivity, and reducing the incidence of false-negative results. Alternatively, the SARS-CoV-2 wastewater surveillance dashboard in Finland denotes inconclusive results to avoid the implications of potential false-negative errors while emphasizing the importance of measures to avoid false-positive errors ( Supplementary Fig. 2 ). Another important factor in data reporting is the lack of pertinent technical information in published articles regarding the sample processing workflow. Such information is crucial for interpreting the potential for false-negative and false-positive errors. Without information regarding the laboratory workflow, it is difficult to determine whether a sub-optimal result was J o u r n a l P r e -p r o o f due to sampling, poor virus concentration and/or RNA extraction efficiency, use of a less sensitive RT-PCR assay, or other factors. When a wastewater surveillance program reports positive results to public health units, they may immediately contact and advise public health agencies and/or community leaders on the location. Local public health units may seek further wastewater testing to confirm the presence of SARS-CoV-2 before actions commence, or they may initiate targeted sampling/testing of localized human populations (i.e., upstream within the sewer systems). Confirmed positive detections lead to public announcements by government agencies, journalists, and others, revealing that SARS-CoV-2 was detected in wastewater. They may also advise a large segment of the population to seek testing for COVID-19 if they live within or have visited the area, and particularly if they have any COVID-19 symptoms. If communitywide clinical surveillance in response to positive wastewater surveillance results fails to identify active infections, the presumed wastewater surveillance false-positive errors may cause policymakers, public health officials, and the public to lose confidence in wastewater surveillance and question its reliability as an early warning system or useful source of information. Conversely, it is possible for COVID-19 infections to be present in the community, and for SARS-CoV-2 RNA to be present in wastewater samples, yet in some cases wastewater sample testing yields negative results (RT-PCR and wastewater surveillance false-negative). Such false-negative errors can be just as problematic as false-positive ones. By failing to alert public health officials to a potential outbreak or by underestimating the extent to which SARS-CoV-2 is circulating in a community, wastewater surveillance false-negative errors may put communities and healthcare workers at unnecessary risk by providing a false sense of security. Also, such false-negative errors without clinical testing may increase in community transmission before any control measures can be put in place, with a concomitant rise in hospital admissions and deaths. This may in turn prompt more severe mitigation measures and subsequent economic impacts than if an outbreak had been detected earlier through wastewater surveillance results. In many communities, the results of SARS-CoV-2 RT-PCR wastewater analyses are published on social media, internet dashboards, and/or in the media. False-negative errors may suggest to the public that SARS-CoV-2 infections are declining or absent in the community, which could result in greater apathy and disregard for public health mitigation measures. In addition, reporting false-negative errors, once discovered, may reduce public confidence in wastewater surveillance. In some jurisdictions, there may be legal consequences for reporting inaccurate results. This has not yet become apparent in practice; however, it should be considered where SARS-CoV-2 RT-PCR screening of wastewater is offered as a service by commercial laboratories. To characterize and mitigate SARS-CoV-2 false-positive and false-negative errors, we recommend consulting the well-established Minimum Information for Publication of Quantitative Real-time PCR experiments (MIQE) guidelines which address data reporting for steps ranging from experimental design to data analysis (Bustin et al., 2009; Huggett et al., 2013) . Nevertheless, such guidelines may need to be adapted to overcome specific As previously discussed, contamination of an RT-PCR assay from extraneous nucleic acid sources is an important potential source of false-positive errors. Nucleic acid from equipment, other samples, and previously synthesized amplicons can contaminate samples or reagents. This section highlights key strategies for identifying and minimizing false-positive errors in wastewater surveillance applications. J o u r n a l P r e -p r o o f Laboratory organization, management, workflow, and QA/QC practices can help minimize the rate of false-positive errors in wastewater surveillance. Thorough cleaning of laboratory work areas with methods proven to destroy or disintegrate RNA on a routine basis (e.g., bleach and UV) is necessary (Huggett et al., 2020) . It is also important to physically separate key steps in the RT-PCR workflow, creating autonomous work areas for sample processing (e.g., pre-treatment and concentration of samples), RNA extraction, RT-PCR master mix set-up (i.e., clean room), amplification, and post-RT-PCR (i.e., gel electrophoresis, sequencing, etc.). Each designated work area should be equipped with dedicated equipment, consumables, and personal protective equipment. Furthermore, cross-contamination between dedicated areas can be eliminated by implementing unidirectional laboratory workflow practices (Millar et al., 2002; Huggett et al., 2020) . Controls should be used to ensure that contamination does not confound results. Contamination during sample collection can be identified by including a field negative control (USEPA, 2014) . A field negative control is performed by dispensing a sample of sterile water into a sample container in the field, which is subsequently transported to the analytical laboratory together with the collected wastewater samples (USEPA, 2002; Esbensen and Ramsey, 2015) . An equipment negative control entails decanting a sample of laboratorygrade water over or through the sampling equipment before collecting a wastewater sample (Esbensen and Ramsey, 2015) . In contrast, a transport negative control is a sample of sterile water that is transported from the laboratory to the sampling site, after which it is retransported to the laboratory without having been exposed to the sampling environment. The transport negative control thus serves to verify that contamination was not introduced due to travel conditions. A method negative control should always be included with each batch of wastewater samples tested to ascertain whether contamination was introduced in the laboratory during sample analysis (USEPA, 2014) . The method negative control should be analyzed in parallel with the wastewater samples using the exact procedure. For the RT-PCR amplification J o u r n a l P r e -p r o o f step(s), multiple no-template controls (NTCs) (minimum of three) should be included with each thermal cycling instrument run. A reverse transcription NTC should also be included for two-step RT-PCR applications to identify whether complementary DNA (cDNA) or SARS-CoV-2 RNA is the source of contamination. It can also be helpful to watch for any spatial patterns in the occurrence of false-positives based on the RT-PCR experiment design, when interpreting results near or below a defined RT-PCR ALOD or ALOQ. Unexpected patterns in contamination detection based on experimental set-up or proximity to samples with a high target concentration or reference materials can suggest sample cross-contamination (Huggett et al., 2020) . Finally, if false-positives are observed in control reactions, it may be useful to sequence these samples to confirm that they are SARS-CoV-2 genetic markers. From the time a wastewater sample is collected from the wastewater system to the time it is processed in the laboratory, it is vital to minimize contact with any materials or aerosols from the field and laboratory environments. The use of probes or any other materials that have come into contact with another sample can provide the opportunity to introduce contamination due to improper cleaning and/or exposure to aerosols. Wastewater samples should be immediately sealed and only opened during the composite preparation, aliquoting, and concentration steps. Practices that reduce the amount of time that samples are exposed to aerosols in the field and laboratory environment are recommended. In addition, it is important to avoid the insertion of any instrument probes into a sample to take measurements, such as temperature or turbidity. Instead, take measurements on a split sample after adequate homogenization and aliquoting. The integrity of new batches of SARS-CoV-2 RT-PCR reagents (i.e., primers, probes, PCR master mix, and DNase/RNase free water) should be confirmed before use (Bustin et al., 2020; Huggett et al., 2020) . This can be achieved by analyzing 10-12 NTC replicates alongside a positive control for each new lot of RT-PCR reagents. Moreover, the required amounts of RT-PCR master mix, primer-probe mix, and nuclease-free water should J o u r n a l P r e -p r o o f preferably be aliquoted for a single use to reduce the potential contamination associated with the frequent use of stock solutions over a prolonged time. SARS-CoV-2 surveillance programs should consider developing a contingency plan to minimize interruptions in laboratory workflow due to contamination. While contamination response plans will likely vary by wastewater surveillance program, there are multiple practices that should be considered when false-positive errors occur (Table 1) . When a falsepositive is observed, it is important to confirm the amplicon by gel-electrophoresis and sequencing. It is also necessary to carefully consider which controls are yielding contamination. For example, if field and method negative controls are negative but the RT-PCR NTC tests identify contamination, then troubleshooting should focus on the amplification work area and methodologies. In addition to verifying the presence of a SARS-CoV-2 falsepositive and the responsible RT-PCR method step, it is necessary to expunge the contaminated reagent or equipment from the sample testing environment. All unsealed samples, laboratory testing materials and reagents used throughout the entire analysis should be discarded. All work area surfaces (e.g., benchtops, floors, etc.) and laboratory equipment (e.g., microcentrifuges, microtube racks, etc.) should be treated with methods proven to degrade nucleic acids. Hydrogen peroxides or ethylene oxide (McEvoy and Rowan, 2019) gas sterilization may be necessary for pipettes and other equipment with working surfaces that are not amendable to surface cleaning. After all work areas and equipment are cleaned, it may be advisable to further confirm that the contamination source has been removed and to implement additional practices to prevent the reoccurrence of contamination. Practices will vary by the source of false-positives; for example, using the NTC contamination example above, it may be useful to use an RT-PCR master mix formulation that includes a deoxynucleotide mix containing uracil instead of thymine along with a heatlabile uracil-N-glycosylase (UNG), which can be used to minimize potential carryover amplificon contamination during the RT-PCR amplification of SARS-CoV-2 (Blanchard et al., 2014; Mascuch et al., 2020) . For other sources of contamination, it may be advantageous to J o u r n a l P r e -p r o o f seek additional analyst training or to implement additional control measures such as equipment or transport negative controls. Finally, it is crucial to demonstrate the absence of contamination leading to false-positive errors by systematically testing field and method negative controls, and RT-qPCR NTCs before returning to wastewater sample testing. Strict enforcement of unidirectional laboratory workflow and dedicated equipment practices. Implement additional contamination monitoring for responsible step. Use different reagent lots Use customized reagents that minimize carryover amplification contamination such as UNG Additional analyst training. Demonstrate absence of contamination Use appropriate controls (e.g., field negative, method negative, and no template) to verify that contamination has been eliminated. Varying challenges associated with different factors can contribute to false-negative errors in wastewater surveillance for SARS-CoV-2, including sampling strategy, variability in community shedding and sewage composition, non-representative sampling, improper sample pre-treatment and storage, lack of laboratory analyst proficiency, and interferences originating from the wastewater sample itself. This section describes practices and potential strategies for identifying and minimizing the occurrence of false-negative errors in SARS-CoV-2 RT-PCR testing. J o u r n a l P r e -p r o o f Practices that limit exposure to suboptimal temperatures during transport and storage before concentration and RNA extraction are recommended. Temperature monitoring systems such as iButton (iButtonLink, Whitewater, WI, USA) can be included during transport to ensure samples are not subject to rapid or long-term fluctuations in temperature, which makes them more susceptible to RNA degradation. This is particularly crucial when sample transportation occurs over multiple days or under extreme temperature conditions. To minimize RNA degradation, samples should be cooled and stored at 4ºC, shipped as quickly as feasible, and processed within 48-72 h after collection (Tan et al., 2018) . Bulk wastewater samples should not be stored at -20ºC and -80ºC to avoid potential damage due to freezing and thawing (Calcott, 1978) . However, concentrated samples that are caked on filters or that are otherwise ready for direct analysis (i.e., ultrafiltered samples) can be stored at -20ºC and -80ºC for longer periods (Salehi and Najafi, 2014) , especially if stabilized in a storage buffer (i.e., guanidinium thiocyanate). RNA extracts can be stored at -20ºC or -80ºC, but multiple freeze-thaws should be avoided. In addition, it is ideal to avoid wastewater sample pasteurization, if possible, because there is currently conflicting information on its effect on SARS-CoV-2 RNA detection. While heat treatment may be necessary to ensure the safety of analysts in sub-optimal laboratory conditions, this hightemperature treatment may reduce the ability to detect trace quantities of SARS-CoV-2 and should be evaluated before practice. Numerous factors can influence the concentration of SARS-CoV-2 in a particular wastewater sample ranging from infection prevalence to individual fecal shedding trends, to the location in the wastewater collection/treatment system where samples are collected. It is therefore important to know and collect as much metadata as possible about the local wastewater system (e.g., relevant COVID-19 public health data from the local community, wastewater flow rate, pre-treatment steps such as chlorination or coagulant addition) to minimize or at least characterize the risk of false-negatives for wastewater surveillance applications. Information on peak flow periods, the impact of local precipitation (e.g., storm events, snow J o u r n a l P r e -p r o o f melts, etc.), and land use in service areas (i.e., residential vs. commercial vs. industrial) can be useful to customize wastewater sampling to maximize the likelihood of detecting SARS-CoV-2 in a community. For example, if a local wastewater facility service area includes industrial and residential sectors, it may be strategic to collect samples from a pump station associated with residential areas rather than collecting a WWTP-influent sample that contains mixed industrial and residential wastewater. In many situations, composite sampling can help minimize false-negatives due to fluctuations in sewage composition over time. This is particularly important for detecting virus shedding from a very few individuals. A flow-weighted composite sample is highly recommended, as it accounts for the fluctuations' inflow at the influent of a WWTP. If this is not possible, a time-weighted composite sample should be the second choice. It may be necessary to conduct pilot experiments to optimize the time intervals and/or flow conditions for optimal composite sampling. If only grab samples are collected in the absence of autosamplers or a lack of logistics, sampling should occur when SARS-CoV-2 loads are expected to be high. It might also be useful to consider collecting multiple grab samples over a short duration (3-4 h) or deploying passive samplers to increase the probability of detecting SARS-CoV-2, especially in areas with low infection rates. During precipitation, larger volumes of wastewater may need to be sampled to compensate for the dilution factor. Analyzing a larger volume (i.e., 1-2 L) will also increase detection sensitivity when COVID-19 cases are low or absent, assuming there is not a corresponding reduction in virus recovery during sample concentration. Primary sludge may be an attractive alternative to untreated wastewater because of solid particles that contain a wide array of enteric viruses Graham et al., 2021) . However, the occurrence of SARS-CoV-2 in primary sludge needs to be further characterized. Any laboratory involved in or intending to participate in wastewater surveillance of SARS-CoV-2 should identify viable long-term options and evaluate virus concentration and RNA extraction methods based on performance and practical aspects (e.g., access to necessary equipment, labor, cost, and potential supply chain limitations) before implementation. SARS-CoV-2 should be concentrated from both liquid and solid phases of wastewater especially J o u r n a l P r e -p r o o f when small volume samples (i.e., <100 mL) of wastewater are processed in regions with low COVID-19 clinical cases. Commercial RNA extraction kits often used in SARS-CoV-2 studies comprise of silica columns specifically designed for the efficient removal of polyphenolic compounds, humic/fulvic acids, tannins, and melanin (La Rosa et al., 2021; Graham et al., 2021) ; however, extractions based on nucleic acid solubility (precipitation) could be more effective in some scenarios. The performance of virus concentration and RNA extraction methods should be determined by seeding an appropriate sample process control in wastewater. If performance is not satisfactory in a series of samples, switching to alternative methods may reduce the likelihood of observing a false-negative due to poor virus concentration and RNA recovery. When possible, it is recommended that RNA recovery be monitored on a samplespecific basis, or at least routinely, due to potential shifts in wastewater composition over time. Systematic errors that introduce unwanted bias in SARS-CoV-2 measurements can lead to false-negative errors. For example, multiple studies have reported substances commonly found in environmental samples that readily bind to nucleic acids and, when bound, force a shift in nucleic acid conformation (Cai et al., 2006) . These interactions have been shown to interfere with nucleic acid recovery in surface waters polluted with wastewater and were undetectable with a molecular process control (Shanks et al., 2016) . It is highly likely that this type of interference can occur in wastewater surveillance. To monitor this potential source of false-negative errors, it is recommended to include a sample process control (i.e., a surrogate virus) to obtain sample-specific information about the recovery efficiency of the entire process or even a specific step within the workflow (Ahmed et al., 2020e; McMinn et al., 2021) . A sample process control should be included for at least 10-20% of the samples tested in a surveillance program. The biological and physical characteristics of the sample process control should be similar to SARS-CoV-2. In the absence of an appropriate surrogate, endogenous controls such as pepper mild mottle virus (Rosario et al., 2009) or crAssphage J o u r n a l P r e -p r o o f (Stachler et al., 2017) can also be used as an indicator of fecal strength or to monitor method performance. Appropriate molecular process controls should be used to monitor the presence of suboptimal RT-PCR assay performance (Hata et al., 2011) and must be included in every wastewater sample tested (Murray et al., 2013) . Pecson et al. (2021) demonstrated that the RT-qPCR methods generally passed regardingtraditional inhibition controls, specifically seeding a molecular process control into a nucleic acid extract and assessing changes in Cq value. However, some samples still exhibited non-sigmoidal amplification curves which might indicate partial inhibition. Therefore, inhibition can be present in multiple ways by preventing the amplification of the intended target or biasing amplification. If inhibitors are suspected, efforts should be made to minimize inhibition to exclude false-negative errors. Multiple strategies exist for mitigating amplification inhibition in PCR-based experiments (Gibson et al., 2012) . In some cases, inhibition can be alleviated by utilizing a different RNA extraction approach capable of removing the specific inhibitor of concern. Others utilize inhibitor-tolerant designer polymerases such as Environmental Master Mix (Thermo Fisher Scientific, Grand Island, NY, USA), which has been shown to reduce the occurrence of amplification inhibition in environmental qPCR applications (Cao et al., 2012) . Another widely applied approach is sample and nucleic acid dilution (Schrader et al., 2012) . While dilution testing is guaranteed to help alleviate inhibition, it also directly reduces the concentration of the genetic target, potentially leading to a false-negative result, and it is not useful when many samples are expected to provide a true-negative results. Therefore, sample or its nucleic acid dilution is not recommended or should be limited (e.g., 2-fold vs. 10-fold) for trace applications such as detecting low levels of SARS-CoV-2 in wastewater. Commercially available nucleic acid clean-up kits can be used to remove PCR inhibitors that are commonly found in complex matrices such as wastewater. J o u r n a l P r e -p r o o f RT-PCR assay protocols, such as those used for surveillance applications, should include optimized annealing temperatures and oligonucleotide concentrations, which may need to be determined empirically in each setting (Bustin et al., 2009; Huggett et al., 2013; Borchardt et al., 2021; Bivins et al., 2021b) . In addition, RT-PCR performance characteristics such as ALOD and ALOQ should be established using appropriate reference materials and following practices outlined in the MIQE guidelines (Bustin et al., 2009; Huggett et al., 2013; dMIQE Group and Huggett, 2020) before wastewater testing. Since the emergence of SARS-CoV-2, mutations and selection pressure on SARS-CoV-2 within the human population have led to new widespread variants in certain regions or worldwide (Peñarrubia et al., 2020) . Although this phenomenon has not yet led to significant issues with the molecular assays commonly used for SARS-CoV-2 detection and quantification, it is important to monitor the genomic properties of local SARS-CoV-2 populations on a routine basis while keeping update on emerging lineages worldwide to allow for timely modification of primer and/or probe sequences to minimize detection biases and, in extreme cases, false-negatives. To reduce the damage to fragile RNA templates, freezing and thawing prior before testing should be avoided. Where possible, it is ideal to test samples immediately after RNA extraction (no freezing before RT-PCR amplification). Material may also be lost due to the absorption of microtubes even when using low-retention plastics. If RNA extracts must be stored for prolonged periods before testing (>30 days), stability should be verified to ensure no degradation has occurred, potentially leading to a false-negative result (The dMIQE group and Hugget, 2020). A molecular processing control could be invaluable providing a reference with a known concentration to determine if the integrity of the RNA extract has significantly changed during storage. It can also be useful to optimize the number of replicates tested to increase the probability of detecting a trace quantity. This practice is important not for wastewater samples and contamination controls (e.g., field and method negative controls, and RT-qPCR NTCs). Increasing the sample volume may also be helpful if it does not result in reduced RNA recovery and/or a higher incidence of RT-PCR amplification inhibition. It is J o u r n a l P r e -p r o o f beneficial to explicitly state and empirically determine ALOD, MLOD, ALOQ and MLOQ definitions. This allows for the unambiguous interpretation of trace detections without any evidence of contamination in control experiments. Finally, it is useful to confirm trace detection in at least a subset of samples via sequencing. Unlike many environmental microbiology research projects which aim to isolate, quantify, or characterize microbiota, the purpose of wastewater surveillance is to inform public health action. Such actions could include large expenditures of resources and the implementation of interventions that are associated with significant impacts on the public. Because of the magnitude of these actions and their potential ramification, wastewater surveillance program data should be of the highest quality. Likely sources of error or bias should be thoroughly explored, and protocols validated such that wastewater surveillance affords accurate information to public health officials. As described herein, this validation should include all aspects of the microbiology workflow from sampling to data analysis. In the absence of such verification, wastewater surveillance is unlikely to afford a reliable source of information. Assess RNA quality and yield before testing. Seed molecular process control to assess extraction efficiency. Validation of extraction method with appropriate surrogate. PCR inhibition Inhibition molecular process control or dilution to assess potential inhibition effects. RT-qPCR or -dPCR amplification Confirm assay sensitivity using local wastewater samples seeded with appropriate control material. Analyse multiple replicates for trace detections. J o u r n a l P r e -p r o o f  Wastewater surveillance is a rapidly developing and growing area of research and is already playing an important role in the COVID-19 pandemic. Wastewater surveillance strategies will also be vital for preventing new outbreaks and managing future pandemics and more traditional endemic pathogens such as seasonal flu, enteric pathogens and antimicrobial-resistant bacteria and their genes.  Many wastewater analysis protocols are not optimized for trace analysis of viruses or other substances, making them susceptible to false-positive and false-negative errors, as discussed in this study. It is important to acknowledge these challenges when interpreting data and inferring the probability that individuals infected with SARS-CoV-2 are present in the community.  Improvements are needed for reliable and rapid detection application. To detect SARS-CoV-2 or any other microbial target of public health interest with greater accuracy, it is essential to set coordinated guidelines on sampling, establish stringent QA/QC practices, and identify effective concentrations, efficient RNA extraction, and highly sensitive and specific RT-PCR assays for detection.  While standardization of some aspects of the laboratory workflow may not be appropriate or possible due to context-specific variability in methods, instruments, and reagents, it is essential to seek broad inter-laboratory harmonization as much as possible.  Additional research is also warranted, including inter-laboratory studies utilizing standardized reference materials and protocols to better understand the variability associated with various aspects of the methods in use. In this way, underperforming laboratories could validate their performance against laboratories that demonstrate superior performance. The views expressed in this article are those of the author(s) and do not necessarily Comparison of concentration methods for quantitative detection of sewage-associated viral markers in environmental waters 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 Decay of SARS-CoV-2 and surrogate murine hepatitis virus RNA in untreated wastewater to inform application in wastewater-based epidemiology Surveillance of SARS-CoV-2 RNA in wastewater: methods optimization and quality control are crucial for generating reliable public health information Comparison of virus concentration methods for the RT-qPCR based recovery of murine hepatitis virus, a surrogate for SARS-CoV-2 from untreated wastewater 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 SARS-CoV-2 RNA monitoring in wastewater as a potential early warning system for COVID-19 transmission in the community Cross-contamination in molecular diagnostic laboratories in low-and middle-income countries: A challenge to COVID-19 testing First confirmed detection of SARS-CoV-2 in untreated municipal and aircraft wastewater in Dubai, UAE: the use of wastewater-based epidemiology as an early warning tool to monitor the prevalence of COVID-19 Environmental surveillance for poliovirus in Haiti (2017-2019): The dynamic process for the establishment and monitoring of sampling sites A recurrent mutation at the position 26340 of SARS-CoV-2 is associated with failure of the E gene quantitative reverse transcription-PCR utilized in a commercial dual-target diagnostic assay Environmental surveillance for polioviruses in the global polio eradication initiative Coronavirus (COVID-19) current situation and case numbers Fate of organic microcontaminants in wastewater treatment and river systems: An uncertainty assessment in view of sampling strategy, and compound consumption rate and degradability Wastewater-based epidemiology to monitor COVID-19 outbreak: present and future diagnostic methods to be in your radar Evaluation of viral concentration methods for SARS-CoV-2 recovery from wastewaters Wastewater-based epidemiology Within-day variability of SARS-CoV-2 RNA in municipal wastewater influent during periods of varying COVID-19 prevalence and positivity Variability in RT-qPCR assay parameters indicates unreliable SARS-CoV-2 RNA quantification for wastewater surveillance Development and validation of a real-time two-step RT-qPCR TaqMan® assay for quantitation of Sacbrood virus (SBV) and its application to a field survey of symptomatic honeybee colonies The Environmental Microbiology Minimum Information (EMMI) Guidelines: qPCR and dPCR quality and reporting for environmental microbiology CoV2-ID, a MIQE-compliant sub-20-min 5-plex RT-PCR assay targeting SARS-CoV-2 for the diagnosis of COVID-19 The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments Interactions of DNA with clay minerals and soil colloidal particles and protection against degradation by DNase Freezing and thawing microbes Effectiveness of qPCR permutations, internal controls and dilution as means for minimizing the impact of inhibition while measuring Enterococcus in environmental waters Droplet digital PCR for simultaneous quantification of general and human-associated fecal indicators for water quality assessment Coronavirus Disease 2019 (COVID-19) CDC 2019-novel coronavirus (2019-nCoV) real-time RT-PCR diagnostic panel for emergency use only instructions for use COVIDView. A weekly surveillance summary of U.S. COVID-19 Activity Approaches applied to detect SARS-CoV-2 in wastewater and perspectives post-COVID-19 SARS-CoV-2, SARS-CoV, and MERS-CoV viral load dynamis, duration of viral shedding, and infectiousness: a systematic review and meta-analysis Time-evolution of SARS-CoV-2 in wastewater during the first pandemic wave of COVID-19 in the metropolitan area of Barcelona Canadian SARS-CoV-2 Interlab consortium. 2021. Comparison of approaches to quantify SARS-CoV-2 in wastewater using RT-qPCR: Results and amplification from a collaborative inter-laboratory study in Canada Molecular diagnosis of a novel coronavirus (2019-nCoV) causing an outbreak of pneumonia Assessing sensitivity and reproducibility of RT-ddPCR and RT-qPCR for the quantification of SARS-CoV-2 in Wastewater Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR Quantitative analysis of SARS-CoV-2 RNA from wastewater solids in communities with low COVID-19 incidence and prevalence An interactive web-based dashboard to track COVID-19 in realtime Data, disease and diplomacy: GISAID's innovative contribution to global health QC of sampling processes -a first overview: from field to test portion Systematic review and meta-analysis of time-temperature pathogen inactivation Sensitivity assessment of droplet digital PCR for SARS-CoV-2 detection An alternative workflow for molecular detection of SARS-CoV-2 -escape from the NA extraction kit-shortage Evaluation of two rapid ultrafiltration-based methods for SARS-CoV-2 concentration from wastewater Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR) Asymptomatic transmission, the Achilles's heel of current strategies to control Covid-19 Early-pandemic wastewater surveillance of SARS-CoV-2 in Southern Nevada: Methodology, occurrence, and incidence/prevalence considerations Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contamination of high-touch surfaces in field settings Measuring and mitigating inhibition during quantitative real-time PCR analysis of viral nucleic acid extracts from large-volume environmental water samples Charting the challenges behind the testing of COVID-19 in developing countries: Nepal as a case study COVID-19 surveillance in Southeastern Virginia using wastewaterbased epidemiology SARS-CoV-2 RNA in wastewater settled solids is associated with COVID-19 cases in a large urban sewershed Comparison of nucleic acid extraction and reverse transcription-qPCR approaches for detection of GI and GII noroviruses in drinking water Wastewater surveillance for SARS-CoV-2: Lessons learnt from recent studies to define future applications First environmental surveillance for the presence of SARS-CoV-2 RNA in wastewater and river water in Japan Wastewater surveillance for SARS-CoV-2 on college campuses: initial efforts, lessons learned and research needs Detection and quantification of SARS-CoV-2 RNA in wastewater and treated effluents: Surveillance of COVID-19 epidemic in the United Arab Emirates Validation of internal controls for extraction and amplification of nucleic acids from enteric viruses in water samples Multi-laboratory survey of qPCR enterococci analysis method performance in U.S. coastal and inland surface waters A novel passive sampling approach for SARS-CoV-2 in wastewater in a Canadian province with low prevalence of COVID-19 Droplet digital RT-PCR to detect SARS-CoV-2 signature mutations of variants of concern in wastewater The detection and stability of the SARS-CoV-2 RNA biomarkers in wastewater influent in Helsinki The digital MIQE Guidelines: Minimum Informantion for publication of quantitative digital PCR experiments Cautionary note on contamination of reagents used for molecular detection of SARS-CoV-2 Evaluation of commercial kits for the extraction and purification of viral nucleic acids from environmental and fecal samples Microbiology of the food chain -Horizontal method for determination of hepatitis A virus and norovirus using real-time RT-PCR -Part 1: Method for quantification Benchmarking virus concentration methods for quantification of SARS-CoV-2 in raw wastewater Minimum information necessary for quantitative real-time PCR experiments Shedding of SARS-CoV-2 in feces and urine and potential role in person-to-person transmission and the environment-based spread of COVID-19 Challenges in Measuring the Recovery of SARS-CoV-2 from Wastewater The architecture of SARS-CoV-2 transcriptome SARS-CoV-2 in wastewater: State of the knowledge and research needs Escherichia coli DNA contamination in AmpliTq Gold polymerase interferes with TaqMan analysis of lacZ Persistence and decay of fecal microbiota in aquatic habitats First proof of the capability of wastewater surveillance for COVID-19 in India through detection of genetic materials of SARS-CoV-2 Surveillance of hepatitis A virus in urban sewages and comparison with cases notified in the course of an outbreak First detection of SARS-CoV-2 in untreated wastewaters in Italy SARS-CoV-2 has been circulating in northern Italy since December 2019: Evidence from environmental monitoring Evaluating recovery, cost, and throughput of different concentration methods for SARS-CoV-2 wastewater-based epidemiology Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study Quantitative SARS-CoV-2 alpha variant B.1.1.7 tracking in wastewater by allele-specific RT-qPCR Primer design for quantitative real-time PCR for the emerging coronavirus SARS-CoV-2 Molecular diagnosos of COVID-19: current situation and trend in China Emergence of SARS-CoV-2 through recombination and strong purifying selection Evaluation of droplet digital PCR for quantification of SARS-CoV-2 virus in discharged COVID-19 patients Optical tracking of the interfacial dynamics of single SARS-CoV-2 pseudoviruses SARS-CoV-2 in wastewater: potential health risk, but also data source Detection of SARS-CoV-2 RNA residue on object surfaces in nucleic acid testing laboratory using droplet digital PCR A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits A review of the most commonly used methods for sample collection in environmental surveillance of poliovirus Terminal sterilization of medical devices using vaporized hydrogen peroxide: a review of current methods and emerging opportunities Development of a Large Volume Concentration Method for Recovery of Coronavirus from Wastewater Presence of SARS-Coronavirus-2 RNA in sewage and correlation with reported COVID-19 prevalence in the early stage of the epidemic in the Netherlands Implementation of environmental surveillance for SARS-CoV-2 virus to support public health decisions: opportunities and challenges SARS-CoV-2 detection by direct rRT-PCR without RNA extraction 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 Risk assessment models and contamination management: implications for broad-range ribosomal DNA PCR as a diagnostic tool in medical bacteriology SARS-CoV-2 in Detroit wastewater Delayed laboratory responses to COVID-19 caused by molecular diagnostic contamination Zika virus persistence in sewage Application of a competitive internal amplification control for the detection of sapoviruses in wastewater Comparative performance of SARS-CoV-2 detection assays using seven different primer-probe sets and one assay kit Estimation of the asymptomatic ratio of novel coronavirus infections (COVID-19) Prevalence of asymptomatic SARS-CoV-2 infection: A narrative review Sampling for PPCPs in wastewater systems: Comparison of different sampling modes and optimization strategies Sampling for pharmaceuticals and personal care products (PPCPs) and illicit drugs in wastewater systems: Are your conclusions valid? A critical review Comparison of digital PCR and quantitative PCR with various SARS-CoV-2 primer-probe sets Measurement of SARS-CoV-2 RNA in wastewater tracks community infection dynamics 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 US Multiple assays in a real-time RT-PCR SARS-CoV-2 panel can mitigate the risk of loss of sensitivity by new genomic variants during the COVID-19 outbreak Comparing analytical methods to detect SARS-CoV-2 in wastewater A comparison of SARS-CoV-2 wastewater concentration methods for environmental surveillance Preliminary results of SARS-CoV-2 detection in sewerage system in Niteroi municipality SARS-CoV-2 RNA titers in wastewater anticipated COVID-19 occurrence in a low prevalence area Prevalence and infectivity of SARS-CoV-2 virus in wastewaters and rivers Pepper mild mottle virus as an indicator of fecal pollution Concentration methods for the quantification of coronavirus and other potentially pandemic enveloped virus from wastewater Occurrence, fate and removal of SARS-CoV-2 in wastewater: current knowledge and future perspectives RNA preservation and stabilization Passive sampling of SARS-CoV-2 for wastewater surveillance PCR inhibitors -occurrence, properties and removal Data acceptance criteria for standardized human-associated fecal source identification quantitative real-time PCR methods Quantitative crAssphage PCR assays for human fecal pollution measurement Wastewater analysis of SARS-CoV-2 as a predictive metric of positivity rate for a major metropolis Sources of variability in methods for processing, storing, and concentrating SARS-CoV-2 in influent from urban wastewater treatment plants ddPCR: a more accurate tool for SARS-CoV-2 detection in low viral load specimens Real-time RT-PCR in COVID-19 detection: issues affecting the results Stability of Zika virus in urine: specimen processing considerations and implications for the detection of RNA targets in urine The ultimate qPCR experiment: producing publication quality, reproducible data the first time Comparison of real time and droplet digital PCR to detect and quantify SARS-CoV-2 RNA in plasma Variability of trace organic chemical concentrations in raw wastewater at three distinct sewershed scales The digital MIQE guidelines update: Minimum information for publication of quantitative digital PCR experiments for 2020 Making waves: Wastewater surveillance of SARS-CoV-2 for populationbased health management Applicability of polyethylene glycol precipitation followed by acid guanidinum thiocyanate phenol-chloroform extraction for the detection of SARS-CoV-2 RNA from municipal wastewater Sampler's Guide: Contract laboratory program guidance for field samplers Sampling guidance for unknown contaminants in drinking water Passive samplers, a powerful tool to detect viruses and bacteria in marine coastal areas Concentration and detection of SARS coronavirus in sewage from Xiao Tang Shan Hospital and the 309 th Hospital Assa'ad A., 2020. Clinical testing for COVID-19 Rapid direct nucleic acid amplification test without RNA extraction for SARS-CoV-2 using a portable PCR thermocycler Correlation of SARS-CoV-2 RNA in wastewater with COVID-19 disease burden in sewersheds Detection of SARS-CoV-2 in raw and treated wastewater in Germany -Suitability for COVID-19 surveillance and potential transmission risks Sewage, salt, silica and SARS-CoV-2 (4S): An economical kit-free method for direct capture of SARS-CoV-2 RNA from wastewater. medRxiv WHO World Health Organization (WHO) list of in-house-developed molecular assays for SARS-CoV-2 detection Guidelines for environmental surveillance of poliovirus circulation Recognizing false positives: synthetic oligonucleotide controls for environmental DNA surveillance Virological assessment of hospitalized patients with COVID-2019 SARS-CoV-2 titers in wastewater are higher than expected from clinical confirmed cases Several forms of SARS-CoV-2 RNA can be detected in wastewaters: implication for wastewater-based epidemiology and risk assessment Characteristics of pediatric SARS-CoV-2 infection and potential evidence for persistent fecal viral shedding Direct RT-qPCR assay for SARS-CoV-2 variants of concern (Alpha, B.1.1.7 and Beta, B.1.351) detection and quantification in wastewater Survivability, partitioning, and recovery of enveloped viruses in untreated municipal wastewater SARS-CoV-2 viral shedding characteristics and potential evidence for the priority for faecal specimen testing in diagnosis Metagenomic analysis of viral nucleic acid extraction methods in respiratory clinical samples Fecal specimen diagnosis 2019 novel coronavirus-infected pneumonia SARS-CoV-2 samples may escape detection because of a single point mutation in the N gene