key: cord-0824115-pcbwby2y
authors: Amereh, Fatemeh; Jahangiri-rad, Mahsa; Mohseni-Bandpei, Anoushiravan; Mohebbi, Seyed Reza; Asadzadeh-Aghdaei, Hamid; Dabiri, Hossein; Eslami, Akbar; Roostaei, Kasra; Aali, Rahim; Hamian, Parisa; Rafiee, Mohammad
title: Association of SARS-CoV-2 presence in sewage with public adherence to precautionary measures and reported COVID-19 prevalence in Tehran
date: 2021-12-23
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2021.152597
sha: 9a34ffe2c27bf158761112403347315fb3a9984c
doc_id: 824115
cord_uid: pcbwby2y

Compared to the growing body of literature on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection and quantification in sewage, there are limited studies reporting on correlations between the viral loads in sewage and the prevalence of infected patients. The present work is a part of the regular monitoring effort for SARS-CoV-2 in wastewater influents from seven wastewater treatment plants (WWTPs) in Tehran, Iran, starting from late September 2020 until early April 2021. These facilities cover ~64% of the metropolis serving >5000,000 M individuals. The study set out to track the trends in the prevalence of COVID-19 in the community using wastewater based epidemiology (WBE) and to investigate whether these measurements correlate with officially reported infections in the population. Composite sewage samples collected over 16 h were enriched by polyethylene glycol precipitation and the corresponding threshold cycle (Ct) profiles for CDC ‘N’ and ‘ORF1ab’ assays were derived through real time RT-qPCR. Monte Carlo simulation model was employed to provide estimates of the disease prevalence in the study area. RNA from SARS-CoV-2 was detectable in 100% (‘N’ assay) and 81% (‘ORF1ab’ assay) of totally 91 sewage samples, with viral loads ranging from 40 to 45,000 gene copies/L. The outbreak of COVID-19 positively correlated (R 2 = 0.80) with the measured viral load in sewage samples. Furthermore, sewage SARS-CoV-2 RNA loads preceded infections in the population by 1 to 2 days, which were in line with public adherence with and support for government instructions to contain the pandemic. Given the transient presence of human host-restricted infections such as SARS-CoV-2, these results provide evidence for assessment of the effectiveness of coordinated efforts that specifically address public health responses based on wastewater-based disease surveillance against not only COVID-19 but also for future infectious outbreaks.

The ongoing coronavirus disease has caused the world to undergo unprecedented changes in a short space of time. According to recent updates (8 September 2021), 222,042,024 confirmed cases and 4,588,950 deaths have been reported globally (CSSE, J., 2021) . Several lines of evidence show that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent for the disease, does not only affect the upper respiratory tract and the lungs but also the ilium and colon, resulting in diverse gastrointestinal manifestations (Amereh et al., 2021; Foladori et al., 2020; Wu et al., 2020a) . A significant number of infected diagnosed patients (1583% of cases) shed viral fragments for a prolonged time, ranging from 14 to 21 days (Lodder and de Roda Husman, 2020; Wu et al., 2020a; Xu et al., 2020) , with 10 2 to 10 8 RNA copies per gram of stool on average (Lescure et al., 2020; Pan et al., 2020; Woelfel et al., 2020) ; and the reported shedding period and magnitude varying greatly among cases. The prolonged incubation time (~5 days) and virus shedding even from asymptomatic individuals allow the virus to spread quickly without medical detection and containment, which subsequently contribute to the development of infection as a whole . The virus, in turn, can be released into wastewater and onsite sanitation systems.

The detection of SARS-CoV-2 RNA in feces and untreated wastewater points towards the potential of wastewater surveillance to capture a near real-time picture of the viral disease burden within a community and also to gain clues on outbreaks within a catchment or a specific region (Wu et al., 2020b; Wurtzer et al., 2020) . The method offers a scalable and cost-effective way to anonymously track population-level infection and disease prevalence  J o u r n a l P r e -p r o o f Journal Pre-proof Coccia, 2020; Keshaviah, 2017; Kumar, 2021) .

Concomitantly with the emergence of COVID-19, several countries -including Australia (Ahmed et al., 2020a) , Spain (Randazzo et al., 2020) , France (Wurtzer et al., 2020) , Sweden (Saguti et al., 2021) , Italy (La Rosa et al., 2020) , India (Kumar et al., 2020b) and Pakistan (Yaqub et al., 2020) -quickly pivoted existing sewage surveillance programs, designed to monitor illicit drugs and other viral pathogens, to follow SARS-CoV-2 by quantifying the virus genetic materials in consecutive wastewater samples. Based on these early successes in detecting and quantifying the novel coronavirus, hundreds of sewage systems all over the worldparticularly in developed nations -began testing their wastewater for SARS-CoV-2 (Gonzalez et al., 2020; Martin et al., 2020; Prado et al., 2020; Torii et al., 2021; Wu et al., 2020b) . However, knowledge about the serious and emerging problem of wastewater surveillance for the presence of SARS-CoV-2 from resource-poor regions and least-developed nations is presently lacking.

Analysis of sewage samples in a municipal wastewater treatment plant (WWTP) in Istanbul, Turkey (Kocamemi et al., 2020) confirmed the presence of novel coronavirus in primary sludge.

Detection of SARS-CoV-2 RNA and its variation along the sewer network has also been previously demonstrated in the vicinity wastewaters of a COVID-19 isolation center in Bangladesh (Ahmed et al., 2021) , reinforcing the use of this approach for community surveillance by wastewater-based epidemiology (WBE) as a new paradigm in public health. It is supported by the limited and cost-intensive testing capacity of these communities as compared with developed resource-rich nations, where the number and proportion of affected cases are mainly determined through individual testing and laboratory-based bio-molecular diagnostics (Abu-Ali et al., 2020; Jeremijenko et al., 2020; Saththasivam et al., 2021) .

On 19 February 2020, Iran reported its first confirmed cases of infections in Qom -a religious temporal variations in virus-specific RNA loadings quantified via WBE in studied WWTPs during the lockdown and implementation of precautionary intervention periods were analyzed to address the practical needs and timelines of concerned authorities and policymakers.

The present study was conducted in Tehran, the second-largest metropolitan area in the Middle East and the most populous city in Iran (Fig. 1) . The city occupies an area of 730 km 2 and encompasses a resident population of about 8.694 million according to the 2016 national census and day-time population of over 12 million because of the massive commute from outlying areas (Statistical Centre of Iran, 2016). Land use is predominantly residential, with some industrial and commercial activity. The climate is semi-arid with an annual mean temperature of 18.5 °C and average annual precipitation of about 220 mm. The city has been highly impacted by the COVID-19 pandemic. The number of daily PCR diagnoses was limited to just about 1,100 in the early months of the outbreak in the city, whereas it reached up to 12,000 during each additional wave of COVID-19.

Tehran's sewage project, as has been defined in the urban master plan, has a collection pipe network of almost 9,000-kilometer-long; in which already 7,000 km is in operation and about 684 × 10 3 cubic meters of sewage is daily collected in the city. So far, just about 64% of Tehran inhabitants (approximately 5.561 million) have access to the public sewerage system while the remaining sewage goes underground or flows over the surface inadvertently creating environmental hazards including surface and groundwater pollution. Nevertheless, the operational performances of treatment plants are satisfactory and meet the basic criteria prescribed by national standards for secondary treatment for discharge into streams. Thus far, the J o u r n a l P r e -p r o o f presence of SARS-CoV-2 in wastewater samples achieved in Iran has been limited (Gholipour et al., 2021; Nasseri et al., 2021; Tanhaei et al., 2021) , including our previous work (Rafiee et al., 2021) . To the best of our knowledge, this is the first study to correlate SARS-CoV-2 RNA loads in wastewater with COVID-19 disease burden in the country. The work is a part of a regular monitoring effort for novel coronavirus in Tehran sanitary sewerage that was initiated on 30 September 2020.

Sewage samples were collected from six medium-sized WWTPs (Shahrak-e Gharb (A), Ekbatan (B), Zargandeh (C), Gheytarieh (D), Sahebgharanieh (E), and Mahllati (F)), all representing urban catchments. Further, Tehran's South wastewater treatment plant (Southern Tehran WWTP) (G) that serves a large part of the metropolitan was also included in the sampling scheme (Table 1) . This plant has been planned with a treatment capacity of 4.2 × 10 6 people. The total number of inhabitants served by these WWTPs, the percent coverage of the total population, and treatment capacity of wastewater have also been summarized in Table 1 . The plants also differed in their connected sewershed area characteristics as well as demographic density. The locations of these plants also are shown in Fig. 1 . About 8 L composite samples of untreated urban wastewater entering each WWTP were collected by sampling wastewater every 60 min in a flow-proportional mode (~500 mL per 1000 m 3 influent wastewater), which was subsequently pooled.

The sampling was performed from late September 2020 until early April 2021 between 8:00 to 24:00, biweekly. The operators of WWTPs at medium-sized units collected the samples, while storing them at approximately 4 °C during sampling; whereas, 24-hour composite samples were collected using an auto-sampler in the large-sized Southern Tehran WWTP. Samples were J o u r n a l P r e -p r o o f transported on melting ice to the laboratory and stored at about 4 °C until further analysis. In total, 13 rounds of samples were taken from 30 th September 2020 to 8 th April 2021. Various physicochemical properties (including BOD 5 , TSS, TN, TP, pH, and temperature) of raw sewage entering each WWTP during the study period were determined. It is worth mentioning that there was no significant rainfall or snowmelt infiltration into the sewer collections systems during sewage sampling.

Samples were immediately concentrated upon receipt in the Research Institute for Gastroenterology and Liver Diseases lab, followed by molecular processing within 24 h of sample collection.

Polyethylene glycol (PEG) precipitation was applied for virus enrichment in sewage samples, as described by Jones and Johns (2009) with modifications outlined previously (Rafiee et al., 2021 ).

An analysis of the data available in the literature already confirms the effectiveness of PEG precipitation for SARS-CoV-2 in sewage and surface water samples (Ahmed et al., 2020b; Haramoto et al., 2020; Torii et al., 2021) . The method began with centrifugation of sewage subsamples (50 mL) at 4000 ×g for 10 min. The top aqueous layer (40 mL) was then recovered carefully and mixed with PEG 8000 50% (w/v) and NaCl (1.5 M). The resultant mixture was incubated overnight at 4 °C (300 rpm) and centrifuged at 15,000 ×g for 30 min at 4 °C. The supernatant was discarded and the resulting pellets were re-suspended in ~300 μL RNase-free water. RNA was directly extracted from this sample.

RNA was extracted from virus concentrates using a QIAamp® Viral RNA mini kit (Qiagen J o u r n a l P r e -p r o o f Journal Pre-proof Company, Hilden, Germany -Catalog No. 52904) following the manufacturer's instructions. All RNA extracts were stored at -80 °C and subjected to reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays for detection of SARS-CoV-2 within 24 h of sample extraction. First, we employed qualitative measurement, and hence, the absence or presence of the virus based on threshold cycles (Ct). Amplification curves were manually inspected using quality control measures and those with threshold cycles beyond 40 were discarded (i.e., Ct=40 was the detection limit). Preliminary testing of samples with ORF1ab and N genes of SARS-CoV-2 as well as RNase P (internal process control) using TaqPath™ performed at 50 °C for 10 min followed by RT inactivation and initial denaturation at 95 °C for 3 min. This was followed by 45 cycles of denaturation at 95 °C for 10 s and annealing/extension at 55 °C for 30 s with a Rotor-Gene Q MDx thermal cycler (QIAGEN Hilden, Germany). Nuclease free water was used as no template control in our study (Sequences of primers and probe was provided in Supplementary information (SI) - Table S1 ).

J o u r n a l P r e -p r o o f Journal Pre-proof All RNA samples obtained from sewage were tested in triplicate reactions and the average quantities were reported in viral gene copies/L ± standard deviation (SD). The assay limit of detection (ALOD, ≥ 95% detection probability) was assessed and determined to be approximately 3 copies/reaction. Likewise, the assay limit of quantification (ALOQ, CV = 35%) was approximately 3.1 copies/reaction. The standard curve used to quantify SARS-CoV-2 in field samples showed a slope, R-squared and primer efficiency value of -3.335, 1 and 0.99, respectively. More precisely, gene copy numbers were calculated as follows, considering the well-established principle of 3.32 Ct change in correspondence to 10-fold change: Gene copy number=10 ((40-Ct)/(10/3.32)) (Kumar et al., 2020a) . Viral concentrations were recorded as gene copies per 1000 mL (gc/L), given the volumes of sample, concentrate (eluate), nucleic acid extracts, and RT-qPCR reaction (Eq. 1).

Virus load (gene copies/L of wastewater) = viral gene copies μL eluted RNA (PCR RNA volume) × 40 μL (total volume of eluted RNA) 50 mL (initial volume of concentrated wastewater sample) × 10 3 (mL/L) (Eq. 1)

To evaluate the efficacy of virus concentration and extraction methods, 15 µL of 4.5 × 10 5 gc of enveloped avian coronavirus (IBV) was seeded into 40 mL of seven wastewater samples from different WWTPs and three 10-fold serial dilutions were tested to determine the method's limit of detection. Positive and negative nucleic acid control extractions of Nuclease-free water with or without the same quantity of IBV spike-in were used to quantify IVB recovery by RT-PCR.

Enrichment, extraction and detection/quantification were all performed as previously outlined.

To obtain a broader picture, the results were used to determine cross-contamination during the nucleic acid extraction process or RT-PCR assay setup and also the effect of sewage on virus recovery. The RNA was isolated from independently processed samples and RT-PCR was J o u r n a l P r e -p r o o f Journal Pre-proof performed. The efficacy of viral recovery was calculated using the following equation (Eq. 2):

2) The mean and standard deviation was calculated.

The prevalence of infected individuals within each sewer catchment was estimated through

Monte Carlo simulation given the total number of SARS-CoV-2 RNA copies in wastewater each day, as measured by RT-qPCR, and the number of viral RNA copies excreted daily in stool by an infected person as the following equation (Eq. 3):

Infected cases = C (gene copies/L) × Q (L/day) Fecal load (g/day/person) × Fecal shedding (viral copies/g stool)

where, C and Q stand for measured viral load in wastewater and corresponding daily influent flow, respectively. Monte Carlo simulation was conducted using the Oracle Crystal Ball (Version 11.1.2.4.850 Oracle©) Excel add-on to account for the variability in some of the factors in Eq. 3.

Per person virus load to the sewage was modeled as a log-uniform distribution from 2.56 to 7.67 by considering the shedding rates of SARS-CoV-2 RNA copies/g of feces during the periods of heaviest shedding among mild cases of COVID-19, as reported elsewhere Wölfel et al., 2020) . Hereby, the daily per capita loads of stool was modelled as a normal distribution with a range of 100-400 g feces/person according to Hart and Halden (2020) and Rose et al. (2015) . It is worth mentioning that the number of viral RNA copies found in stool varies from patient to patient and varies within a single patient as the disease progresses.

According to Li et al., (2021) , from pooled stool samples, the mean shedding magnitude was about 10 4.52 copies/g with a 95% confident interval ranging from 10 4.26 to 10 4.78 copies/g. The daily flow rates of wastewater were provided by Tehran sewerage Co. and the average per capita wastewater rate was calculated as of 220 L/person/day. Likewise, published census data coupled J o u r n a l P r e -p r o o f with WWTPs design capacities were used to estimate the inhabitant population of each catchment area.

To improve the accuracy of the prediction model in estimating the number of people infected, the viral load shed per mL of urine in infected persons, as well as the recovery percentage of viral particles in wastewater was added. The urinary viral load was taken to be 2.50 Log 10 per mL (Peng et al. 2020) . The daily volume of urine per capita was modeled as a log-uniform distribution with a minimum of 2.78 and a maximum of 3.76 (Lemann Jr et al., 1996) .

In the present study, recovery efficiency was 42.55 ± 12.45% of IVB spiked into untreated wastewater. Therefore, the recovery efficiency was modeled as a uniform distribution with a minimum recovery of 30.1% and a maximum of 55%. The revised model was employed to estimate the total number of infected people through the following equation (Eq. 4): It is worth noting here that along with some of the uncertainties touched upon above, RNA losses in the sewer may undermine the accuracy of prevalence back-estimation through WBE. Despite the transient infectivity of SARS-CoV-2 in wastewater environment, some have argued that its RNA is significantly more persistent, reaching 3-33 days (Wu et al., 2020b) . Owing to the lack of sufficient information, the RNA losses in the sewer were ignored in our study, knowing that this approach can result in an underestimation of the infected population (Kapo et al., 2017; Weidhaas et al., 2021) . In addition to the fact that only 64 % of the inhabitants have access to the (Amereh et al., 2021; Corman et al., 2020) . The 'ORF1ab' gene assay has also been considered as confirmatory testing (specific to SARS-CoV-2) in some studies to estimate the infection incidence within the population. The expression of 'ORF1ab' requires ribosomal frame shifting, implying that it is produced at significantly lower levels as compared to N-encoded functions and sub-genomic RNA (Barra et al., 2020) . Therefore, in the infected samples, the 'ORF1ab' copy number is expected to be lower than 'N'. In contrast, the 'N' target is highly expressed because its sequence is present in almost all sub-genomic RNA (Barra et al., 2020; Kim et al., 2020) .

The method (in terms of concentration, extraction and detection) used herein showed a 42.55 ±12.45% % (average and standard deviation) recovery efficiency for IVB, a surrogate for SARS-CoV-2 from untreated wastewater. This recovery was comparable to those reported by Ahmed et al. (2020c) and outperformed others demonstrating 26.7 ± 15.3% and 31 to 32% recovery efficiencies for the murine hepatitis virus from wastewater (Ahmed et al., 2020b) and human adenovirus from river water (Ahmed et al., 2015) , respectively. PEG precipitation seems to be an efficient method to recover SARS-CoV-2 RNA from various environmental matrices and has previously been used for the enrichment of viruses from aqueous solutions ( reported a 1-1.5 log 10 reduction in detection following PEG precipitation and RNA isolation.

Therefore, the presence of inhibitors in wastewater samples needs to be investigated, and if present, efforts should be taken to minimize overall inhibition. To achieve this, it is recommend that each wastewater sample be seeded with a surrogate virus as a whole-process control to obtain information on the surrogate virus and RNA recovery as well as RT-PCR inhibition for the entire process starting from sample concentration to RT-PCR detection. Accordingly, we used IBV as a whole process control in our study. This method, however, has been reported to provide improved recovery of viral particulates compared to ultrafiltration and other methods (Ahmed, 2020c (VSV)) to SARS-CoV-2 in structure and morphology. To date, a detailed comparison between different surrogate viruses and SARS-CoV-2 in the concentration, extraction and detection from wastewater is still lacking, which requires further investigations (Kantor et al., 2021) . Subsequently, variation in the viral recoveries can represent not only differences in the concentration methods but also in the structure, genetic components, as well as the behavior of different surrogate viruses.

A total of 91wastewater samples were collected every 2 weeks from seven major municipal WWTPs (cumulative inlet loading > 1. and 300 gene copies/mL in sewage samples (Wu et al., 2020a) . Likewise, Wurtzer et al. (2020) reported viral loads as high as 3.2 × 10 6 genome units/L in Paris (France), which is the highest SARS-CoV-2 concentration ever reported in wastewater influent. They also reported a significant positive association between SARS-CoV-2 concentrations in WWTP influents and the number of infected individuals in their study region. It is important to caveat a lack of standardization in calculating from copies per reaction based on a standard curve to gene copies/L of SARS-CoV-2 which may significantly impact the range of values reported, and therefore comparisons. However, it is encouraging to compare these findings with those reported for a few Middle East countries like Turkey (Kocamemi et al., 2020) , Qatar (Saththasivam et al., 2021) , United Arab Emirates (Hasan et al., 2021) , and Pakistan (Yaqub et al., 2020) , whose J o u r n a l P r e -p r o o f wastewater viral concentrations have been published. We recognize stark distinction between SARS-CoV-2 titers in our study and what other communities, in particular countries in the region, are reporting. Differences between studies are also abundant, and can include variability in the severity and load of fecal shedding, the efficiency of RNA recovery from sewage samples, and RNA losses in sewage pipes and during experimental procedures, among others. To verify the above viewpoints, the maximum SARS-CoV-2 RNA concentrations reported in raw sewage samples ranged from 1.2 × 10 3 to 3.2 × 10 6 gene copies/L (Ahmed et al., 2020a; La Rosa et al., 2020; Nemudryi et al., 2020; Randazzo et al., 2020; Wurtzer et al., 2020) . corresponding viral loads throughout the 29-week study period are comparable among WWTPs (Fig. 3) , with an increase in SARS-CoV-2 viral load from mid-October. Ct values decreased to < 31.27 (Fig. 4) , which coincides with the population-wide COVID-19 resurgence as observed in sites. This inconsistency may be explained by the rather discrete location of Sahebgharanieh catchment, which is located in the north of the city and is characterized as a built up area of the city with relatively low population density. One of the benefits that emerges from wastewater testing and in particular the spatial variations of viral load numbers is that tracking large-scale infectious disease through sewage analyses offers a stylized view of the main workings of the epidemic than the total number of infected individuals in the community. This is particularly important in light of the variability in the severity and shedding duration among the population (Saguti et al., 2021; Hasan et al., 2021) . Taken together, our results confirm that SARs-CoV-2 concentrations in wastewater closely coincide with or precede confirmed virological laboratory testing, as has been previously reported (Medema et al., 2020; Saguti et al., 2021; Trottier et al., 2020; Wurtzer et al., 2020) .

Several studies have reported that the quantity of SARS-CoV-2 in a sewer catchment is correlated with the actual number of infected individuals in that sampled area (Fongaro et al., 2021; Kitamura et al., 2021; Saththasivam et al., 2021) . Herein, viral load numbers in sewage samples were converted to the number of infections based on the analysis of Monte Carlo simulation to explore whether these measurements mirror infections in the population.

Taking into account the normal distribution of cases, it is only possible to compare the daily J o u r n a l P r e -p r o o f positive cases against the total estimated infected population of the studied WWTPs. It is important to bear in mind that the city is served in part (64%) by these sewage works. outbreak monitoring very recently using a similar approach in Qatar (Saththasivam et al., 2021) .

In line with a generally assumed long incubation period of SARS-CoV-2 (Li et al., 2021) , the number of infections on any given date was roughly calculated by taking the sum of officially declared confirmed cases on the day of sampling, 10 days before the sampling date, and 11 days following each sampling date. It implies that the summation did account for the patients already (Fig. 5) . Overall, the estimated number of individuals shedding SARS-CoV-2 within sewersheds was found to be linearly correlated with the cumulative diagnosed positive cases (R 2 = 0.80, p < 0.001) (Fig. 6) .

Moreover, it can be inferred that the estimated infected population against the officially declared case counts is 353:1. More specifically, the estimated sum of the infected population is more than the sum of the confirmed cases during the time course of sewage monitoring. While theoretically it is expected to be a 1:1 ratio, this result is corroborated by prior research (Albastaki et al., 2021; Wu et al., 2021) and could be attributed to the inclusion of both the diagnosed and undiagnosed cases in sewage viral loads. La Rosa et al. (2020) speculated that a substantial number of the population are either asymptomatic or paucisymptomatic and would otherwise remain clinically undetected. Likewise, earlier studies also noted a lack of symptoms in up to 70 % of infections (Byambasuren et al., 2021; Oran and Topol, 2021) . Furthermore, Meyerowitz et al. (2020) found a range of pre-symptomatic or asymptomatic COVID-19 cases averaging 43 ± 28%. As yet, limited parallel research has definitely established strong correlations between viral loads in sewage and the prevalence of clinically positive patients.

While variable correlations do exist in the global literature ranging from 0.18-0.81 Randazzo et al., 2020; Weidhaas et al., 2021) . However, it is important to note that correlations with wastewater are typically carried out based on populations of individuals which test positive on a given day, and largely ignores the fact that mass testing is not carried out frequently. Therefore, positive cases typically represent the proportion of the population which J o u r n a l P r e -p r o o f has symptoms and ignores large societal inequality and variabilities which impact testing including availability of testing, affordability, personal biases and opinions, institutional distrust, etc. More studies of comparable study populations are thus needed to address which associations remain consistent.

An important limitation of this study is that the exact population being served by each WWTP is not known. 

Despite international efforts to contain SARS-CoV-2 through the implementation of various J o u r n a l P r e -p r o o f regional and continent specific policies, it is still posing a serious global challenge. The long incubation period, viral shedding and transmission by asymptomatic and pre-symptomatic infected individuals, and virus mutation add to the difficulties in managing the outbreak.

Wastewater-based disease surveillance, as a new paradigm in public health, has been suggested for monitoring community outbreaks; however, comparably little is known about the interrelationships between wastewater viral titers and the abundance of recorded clinical cases within the community, although this has greatly improved over ~21 months since the pandemic began. Various methodological challenges associated with WBE would affect the accuracy of prevalence estimation. To date, the overall uncertainty of WBE and the impact of each step on the prevalence estimation are largely unknown. In the other words, the differences between the estimated number of cases infected by SARS-CoV-2 (based on the viral load of wastewater) and

the officially reported cases of COVID-19 may be the result not only of the lack of a more extensive laboratory diagnosis but also of the uncertainties considered in this work within the model for estimating the number of infected people.

Wastewater viral titers were found to be linearly correlated with the diagnosed caseloads, however, considerable differences were observed between the estimated SARS-CoV-2 shedders and officially reported cases of COVID-19. As mass testing is not a thing, this is literally something that affects every WBE study. Overall, this study reiterates the effectiveness of precautionary efforts in the fight against not only COVID-19 but also future infectious outbreaks using wastewater analysis. 

Tracking SARS-CoV-2 RNA through the wastewater treatment process

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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

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

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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

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Analytical sensitivity and specificity of two RT-qPCR protocols for SARS-CoV-2 detection performed in an automated workflow

Wastewater-based epidemiology: global collaborative to maximize contributions in the fight against COVID-19

Comparison of seroprevalence of SARS-CoV-2 infections with cumulative and imputed COVID-19 cases: systematic review

CDC. 2019-Novel Coronavirus (2019-nCoV) real-time rRT-PCR panel primers and probes. 2020

Factors determining the diffusion of COVID-19 and suggested strategy to prevent future accelerated viral infectivity similar to COVID

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

COVID-19 dashboard by the

SARS-CoV-2 from faeces to wastewater treatment: what do we know? A review

The presence of SARS-CoV-2 RNA in human sewage in Santa Catarina, Brazil

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Evaluation of pepper mild mottle virus as an indicator of human faecal pollution in shellfish and growing waters

First environmental surveillance for the presence of SARS-CoV-2 RNA in wastewater and river water in Japan

Computational analysis of SARS-CoV-2/COVID-19 surveillance by wastewater-based epidemiology locally and globally: Feasibility, economy, opportunities and challenges

Detection and quantification of SARS-CoV-2 RNA in wastewater and treated effluents: Surveillance of COVID-19 epidemic in the United Arab Emirates

Monitoring SARS-CoV-2 in municipal wastewater to evaluate the success of lockdown measures for controlling COVID-19 in the UK

Population and housing censuses

Evidence for and level of herd immunity against SARS-CoV-2 infection: the ten-community study

Improved detection of F-specific RNA coliphages in fecal material by extraction and polyethylene glycol precipitation

Real-time estimation of the risk of death from novel coronavirus (COVID-19) infection: inference using exported cases

Challenges in Measuring the Recovery of SARS-CoV-2 from Wastewater

Estimation of US sewer residence time distributions for national-scale risk assessment of down-the-drain chemicals

The potential of wastewater testing for public health and safety

Insufficient sensitivity of RNA dependent RNA polymerase gene of SARS-CoV-2 viral genome as confirmatory test using Korean COVID-19 cases

Efficient detection of SARS-CoV-2 RNA in the solid fraction of wastewater

Routine SARS-CoV-2 wastewater surveillance results in Turkey to follow Covid-19 outbreak

Prevalence of SARS-CoV-2 in communities through wastewater surveillance-a potential approach for estimation of disease burden

First proof of the capability of wastewater surveillance for COVID-19 in India through detection of genetic material of SARS-CoV-2

First proof of the capability of wastewater surveillance for COVID-19 in India through detection of genetic material of SARS-CoV-2. Science of The Total Environment

First detection of SARS-CoV-2 in untreated wastewaters in Italy

Urinary oxalate excretion increases with body size and decreases with increasing dietary calcium intake among healthy adults

Clinical and virological data of the first cases of COVID-19 in Europe: a case series

Virus shedding dynamics in asymptomatic and mildly symptomatic patients infected with SARS-CoV-2

Uncertainties in estimating SARS-CoV-2 prevalence by wastewaterbased epidemiology

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

Tracking SARS-CoV-2 in sewage: evidence of changes in virus variant predominance during COVID-19 pandemic

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

Towards an accurate and systematic characterisation of persistently asymptomatic infection with SARS-CoV-2. The Lancet Infectious Diseases

Recovery of diverse microbes in high turbidity surface water samples using dead-end ultrafiltration

The presence of SARS-CoV-2 in raw and treated wastewater in 3 cities of Iran: Tehran, Qom and Anzali during coronavirus disease 2019 (COVID-19) outbreak

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

The proportion of SARS-CoV-2 infections that are asymptomatic: a systematic review

Viral load of SARS-CoV-2 in clinical samples

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

Preliminary results of SARS-CoV-2 detection in sewerage system in Niterói municipality

Moore swab performs equal to composite and outperforms grab sampling for SARS-CoV-2 monitoring in wastewater

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

Presence and infectivity of SARS-CoV-2 virus in wastewaters and rivers

The characterization of feces and urine: a review of the literature to inform advanced treatment technology

Surveillance of wastewater revealed peaks of SARS-CoV-2 preceding those of hospitalized patients with COVID-19

COVID-19 (SARS-CoV-2) outbreak monitoring using wastewater-based epidemiology in Qatar

First detection of SARS-CoV-2 RNA in wastewater in North America: a study in Louisiana, USA

The first detection of SARS-CoV-2 RNA in the wastewater of Tehran

Applicability of polyethylene glycol precipitation followed by acid guanidinium thiocyanate-phenol-chloroform extraction for the detection of SARS-CoV-2 RNA from municipal wastewater

Post-lockdown detection of SARS-CoV-2 RNA in the wastewater of

Correlation of SARS-CoV-2 RNA in wastewater with COVID-19 disease burden in sewersheds

Clinical presentation and virological assessment of hospitalized cases of coronavirus disease 2019 in a travel-associated transmission cluster

SARS-CoV-2 titers in wastewater foreshadow dynamics and clinical presentation of new COVID-19 cases

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This work was supported by National Institute for Medical Research Development (NIMAD) under Grant No. 994141. We would like to acknowledge Tehran Sewerage Co. for assistance J o u r n a l P r e -p r o o f Journal Pre-proof with sample collection. We would also like to acknowledge Research Institute for Gastroenterology and Liver Diseases that generously provided us with their laboratories. The authors also thank anonymous reviewers for their valuable comments on this manuscript.

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