key: cord-0985748-gu5umkiu
authors: Saththasivam, Jayaprakash; El-Malah, Shimaa S.; Gomez, Tricia A.; Jabbar, Khadeeja Abdul; Remanan, Reshma; Arun, K. K.; Ogunbiyi, Oluwaseun; Rasool, Kashif; Ashhab, Sahel; Rashkeev, Sergey; Bensaad, Meryem; Ahmed, Ayeda A.; Mohamoud, Yasmin A.; Malek, Joel A.; Abu Raddad, Laith J.; Jeremijenko, Andrew; Abu Halaweh, Hussein A.; Lawler, Jenny; Mahmoud, Khaled A.
title: COVID-19 (SARS-CoV-2) Outbreak Monitoring using Wastewater-Based Epidemiology in Qatar
date: 2021-02-09
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2021.145608
sha: f0f463d96637496e376f4f02aac46338db9a0584
doc_id: 985748
cord_uid: gu5umkiu

Raw municipal wastewater from five wastewater treatment plants representing the vast majority of the Qatar population was sampled between the third week of June 2020 and the end of August 2020, during the period of declining cases after the peak of the first wave of infection in May 2020. The N1 region of the SARS-CoV-2 genome was used to quantify the viral load in the wastewater using RT-qPCR. The trend in Ct values in the wastewater samples mirrored the number of new daily positive cases officially reported for the country, confirmed by RT-qPCR testing of naso-pharyngeal swabs. SARS-CoV-2 RNA was detected in 100% of the influent wastewater samples (7,889 ± 1,421 copy/L – 542,056 ± 25,775 copy/L, based on the N1 assay). A mathematical model for wastewater-based epidemiology was developed and used to estimate the number of people in the population infected with COVID-19 from the N1 Ct values in the wastewater samples. The estimated number of infected people in the population on any given day using the wastewater-based epidemiology approach declined from 542,313 ± 51,159 to 31,181 ± 3,081 over the course of the sampling period, which was significantly higher than the officially reported numbers. However, seroprevalence data from Qatar indicates that diagnosed infections represented only about 10% of actual cases. The model estimates were lower than the corrected numbers based on application of a static diagnosis ratio of 10% to the RT-qPCR identified cases, which is assumed to be due to the difficulty in quantifying RNA losses as a model term. However, these results indicate that the presented WBE modelling approach allows for realistic assessment of incidence trend in a given population, with a more reliable estimation of the number of infected people in a population at any given point in time than can be achieved using human biomonitoring alone.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has given rise to more than 91.6 million laboratory-confirmed infections and a large number of Coronavirus Disease cases, in addition to over a million deaths globally as of January 2021 (World Health Organization, 2020) . Qatar is an Arab country with an estimated population of 2.8 million people (Planning and Statistics Authority of Qatar, 2020). The first SARS-CoV-2 community transmission case was recorded on March 6, 2020 with the source of infection being traced to a shared-accommodation setting of migrant workers (Abu-Raddad et al., 2020a; Kuwari et al., 2020) . By October 29, 2020, 965,389 people had undergone RT-qPCR testing (34% of the population), with an infection rate that exceeded 50,000 laboratory-confirmed infections per million population, one of the highest worldwide (Abu-Raddad et al., 2020b; Jeremijenko et al., 2020) . The positivity rate among those tested reached a high of 35.4% at the epidemic peak towards the end of May 2020 (Abu-Raddad et al., 2020a) . Since then, the epidemic declined rapidly at first before plateauing for several weeks at a constant incidence (Abu-Raddad et al., 2020a) .

A national population-based survey was conducted in the first week of May 2020 and estimated the weighted SARS-CoV-2 RT-qPCR prevalence (for the total population of Qatar) at importance to identify the emergence of a second wave or surge in Qatar due to the young age profile of the population with only 2% being >60 years of age, the sociodemographic and other factors that contribute to asymptomatic and mild disease. During the first wave in Qatar, communities were identified where less than 15% of the antibody positive subjects had a prior PCR positive diagnosis of SARS-CoV-2 infection. This indicates that there was significant unidentified spread of SARS-CoV-2 in Qatar during the first wave.

Wastewater-Based Epidemiology (WBE) has been shown to have excellent potential for early detection of the outbreak of diseases and to be a useful tool for monitoring biomarkers of human health at the population level. Wastewater contains a diverse range of chemical and biological targets which gives incredibly detailed information about the population that contributes to it. However, the successful extraction from the matrix and subsequent analysis of specific targets can prove to be difficult. Many countries have taken appropriate steps in investing in WBE practice (Asghar et al., 2014; Feng et al., 2018) . A working strategy aims for effective monitoring where WBE can help to detect and estimate the number of infected cases, followed by clinical testing to identify the affected people.

Many WBE studies have confirmed the detection of SAR-COV-2 in the influent of wastewater treatment plants and its significant correlation with the reported COVID-19 cases Medema et al., 2020) . Viral loads ranging between 7.50E+02 and 3.40E+04 gene copies/L were detected in the influent of several wastewater treatment plants in the United Arab Emirates (Hasan et al., 2020) . Another WBE study conducted in the region of Murcia (Spain) reported that 35 out of 42 of wastewater influent samples were positive while all the tertiary water samples were tested negative (Randazzo et al., 2020) . Analyses of wastewater J o u r n a l P r e -p r o o f Journal Pre-proof samples collected from a wastewater plant in Ahmedabad, Gujarat, India indicated that all the samples were tested positive with an estimated maximum concentration of 3500 copies/L. Viral RNA is found in nasal discharge, sputum, blood or feces (Huang et al., 2020) or urine . There are many reports of SARS-CoV-2 shedding in the human stool that have been documented in recent literature Holshue et al., 2020) . Using a WBE modelling approach, the number of people in the population infected with SARS-CoV-2 can be estimated based on:-(i) concentration of SARS-CoV-2 RNA at the inlet of the wastewater treatment plant, (ii) volumetric flow rate of the wastewater treatment plant, (iii) fecal load, (iv) RNA shedding in the stool, and (v) RNA losses in the sewer pipe. The Fecal Load is the amount of feces generated per person. The reported range in the literature is 51-796 g feces/day/person at an average density of 1.06 g/mL (Hart and Halden, 2020; Rose et al., 2015) . The RNA fecal shedding represents the number of RNA copies found in one gram of feces. The amount of RNA found in stool varies from patient to patient and varies within a single patient as the illness progresses. It has been reported that range (1,000-10,000,000) RNA copies of SARS-CoV-2 can be found in a gram of feces, with a peak more than 10 7 during the first week of the signs and symptoms of COVID-19 (Wölfel et al., 2020) . Another study reported a median viral load of 10 5.1 and 10 3.9 copies/mL in COVID-19 patients with and without diarrhea respectively (Cheung et al., 2020) . The RNA loss factor accounts for losses due to the degradation of the viral RNA over time, adsorption onto solids, and other factors related to losses during transit. Hart and Halden (2020) stressed the importance of incorporating temperature effect and sewer transit time in WBE models, as these factors govern the decay kinetics of SARS-CoV-2.

In order for COVID-19 WBE to provide a reliable early warning system that is fit for purpose, RNA recovery and detection methods must also be sensitive enough to quantify low J o u r n a l P r e -p r o o f Journal Pre-proof viral concentrations in a wastewater sample. Several extraction methods have been reported in the literature such as centrifugal ultrafiltration (Medema et al., 2020) , adsorption-extraction method using electronegative membranes Sherchan et al., 2020) , aluminum hydroxide adsorption-precipitation method (Randazzo et al., 2020) , and PEG concentration method with or without ultrafiltration Alpaslan Kocamemi et al., 2020; Kumar et al., 2020; Wu et al., 2020) ; the glycine buffered with PEG extraction concentration protocol was applied in this study. Murine hepatitis virus (MHV) enveloped virus was examined in raw wastewater samples, with a recovery rate ranging between 26.7 and 65.7% . However, the recovery efficiency of enveloped SARS-CoV-2 may be different from that of other enveloped and non-enveloped enteric viruses .

The total population served by the wastewater treatment plants (WWTPs) can be estimated by using published census data, WWTP design capacity, biomarkers, and/or the water quality parameters of wastewater. In this study, ammonium-nitrogen (NH 4 + -N), an indirect biomarker of urine, is used to estimate the total population served by each WWTP. Despite these challenges, this WBE approach coupled with the data generated can be broadly applied globally is significantly less expensive than large scale human biomonitoring campaigns (Hart and Halden, 2020) . This would help in managing the pandemic and accelerating the global economic recovery.

Here, we report, for the first time, the presence of SARS-CoV-2 RNA fragments in municipal wastewater in the State of Qatar and we present an estimation of the number of infected people in the population by applying a WBE model. The WBE estimated numbers were found higher than the reported daily cases identified by clinical testing and indicated reasonable agreement with estimated numbers using a seroprevalence calculated clinical diagnosis ratio. 

Samples were processed using the PEG extraction protocol as described previously Bibby and Peccia, 2013) . Briefly, 25 mL of glycine buffer (0.05 M glycine, 3% Bovine Serum Albumin (BSA)) was added to 200 mL of wastewater to detach the virions particles from the organic matter, which binds to organic material. The mixture was incubated at 4˚C for 2 hours with shaking at 200 rpm. The samples were then centrifuged at 4,500×g for 30 min at 4°C (without brake) to remove large debris and bacterial cells. After

Journal Pre-proof centrifugation, the supernatant was carefully transferred into a sterilized container. PEG 8000 (100 g/L) and NaCl (22.5 g/L) was added to the supernatant and dissolved by gentle shaking to avoid warming up the solution. The dissolved mixture was incubated overnight at 4°C and 100 rpm agitation followed by centrifugation for 90 min (13,000×g and 4 °C) without braking. After centrifugation, the sample was decanted carefully and the sample was centrifuged again for 10 min at 13,000×g 4 °C with brake set to 3 (of 9). The resulting viral-containing pellet was eluted in 1 mL DNA/RNA Shield™ (Zymo Research, Irvine CA, USA Cat. No. R1100-250) and stored at -80°C until further processing. All of the samples were processed in duplicate except for the July 19, 2020 batch which was processed in triplicate.

Viral RNA was extracted from 200 μL of the concentrated wastewater sample using Quick-RNA Viral Kits (Zymo Research, Irvine CA, USA Cat. No. R1041) according to the manufacturer's protocol. The viral RNA was eluted in 30 µL of nuclease-free water and stored at -80°C until the next step. All samples were extracted in duplicate.

The detection of SARS-CoV-2 in the extracted viral RNA was performed using SARS- 2020) . Table 1 Quencher (none). All RT-qPCR assays were duplicated.

Positive controls were used for RNA extraction. The control for RNA extractions were conducted on 4 batches collected in July 2020 by running RT-qPCR using the human RNAP gene assay from the CDC set of assays (Table S1 ). This control is based on the assumption that wastewater will contain a certain amount of human RNA and allow us to ensure the full process did not regularly introduce inhibitors of RNA extraction or RT-qPCR. Any human RNA present in the wastewater goes through the full extraction process and positive results ensure that RNA extraction occurred sufficiently to allow viral RNA detection.

J o u r n a l P r e -p r o o f The amplification efficiencies (E) were calculated based on the equation: E=10 (−1/slope) −1.

Negative and positive controls were included in each qPCR run and all qPCR assays were Table 2 . Equation (1) with the mean values of the parameters in the denominator to obtain an estimate of the infected population. However, because of the unavoidable variations, it is impossible to calculate the exact value of N; there will be a range of N values that can all lead to the measured RNA concentration. This range can be calculated using a statistical Monte-Carlo-Bayesian approach that is explained in detail in the supporting information. Importantly, the central limit theorem of probability theory allows us to avoid performing lengthy Monte-Carlo calculations and instead apply a simple formula to calculate the standard deviation in the infected population δN. The calculated value is expected to be accurate whenever the formula gives a value for δN that is significantly smaller than N.

In our study, the sewer transit time is highly variable as some of the WWTPs are also served by tankers that transport wastewater from the districts and areas that are not connected to the sewage network. The determination of RNA losses in wastewater samples due to the myriad of factors listed earlier (e.g., adsorption, temperature, half-life, etc.) is difficult to estimate. Due to a lack of sufficient information, the RNA losses in the sewer were ignored (γ = 0) in this study, with the understanding that this approach can lead to an underestimation of the infected population. The total population served by the WWTPs was calculated based on Equation (2) using NH 4 + -N as a biomarker.

As the infected population excretes SARS-CoV-2 virus over a period of time which varies from person to person, the measured C RNA at the inlet a WWTP represents a cumulative concentration shed by patients that were infected several days or weeks before each sampling day. In this study, a cumulative estimate of 22 days is used as a reference based on a clinical study that reported the median duration of SARS-CoV-2 virus in the stool of infected patients as 22 days with an interquartile range between 17-31 days (Zheng et al., 2020) . Assuming a typical (Zheng et al., 2020) # calculated based on the data reported by Zheng et al. (2020) 

The SARS-CoV-2 RNA Nucleocapsid (N) genes were successfully amplified in the raw wastewater composite samples, where all the samples collected from the five major WWTPs in Qatar were SARS-CoV-2 positive with RT-qPCR assays using the Nucleocapsid (N) target gene (N1 and N2) assays. To minimize the human health risk during sample processing inside the laboratory, the collected samples were inactivated by heat exposure at the collection site. Heat pasteurization of two composite samples for 30 and 60 min showed comparable average Ct values for N1 and N2, indicating minimal variation in the viral RNA integrity between the two treatment times ( Figure 1A ). WHO have recommended the heat treatment of 56 °C for SARS-CoV-1 virus inactivation (World Health Organization, 2003) , and a 30-min heat inactivation at 60 °C was sufficient for a greater than 6 log decrease in SARS-CoV-1 viral infectivity in wastewater samples (Rabenau et al., 2005) . At 56°C, no infectious virus was detected within 30 min in nasopharyngeal and sera samples containing SARS-CoV-2 (Batéjat et al., 2020) . Pastorino et al. (2020) reported that 56 °C for 30 min and 60 °C for 60 min did not affect significantly the number of detectable RNA copies in the clinical samples. Based on these results

Journal Pre-proof we identified that 30 min is sufficient for safety purposes without a significant loss in the viral RNA integrity.

The results of the RT-qPCR assay are consistent for both 2019-nCOV_N1 and 2019-nCOV_N2 assays. All RT-qPCR samples were positive and within the quantification level (Ct values below 35 cycles). As shown in Figure 1B (Table S1) revealed that the RNAP Ct values were relatively constant in the samples tested here and so may serve well for comparing results across seasons, population sizes contributing to a collection and different countries.

SARS-CoV-2 RNA (N1 and N2 assays) was detected in all influent wastewater samples (n = 43) collected from the five major WWTPs in Qatar (cumulative influent loading > 760,000 m 3 of wastewater per day) over the sampling period, between 21 st June 2020 and 30 th August 2020. The viral loads in all samples collected from 5 WWTPs during this period can be seen in Table S2 . As shown in Figure 2 , except for WWTP 1 all locations started to witness a decrease in SARS-CoV-2 viral load from late June. In spite of the initial increase of viral load for WWTP1 area until 12 th July 2020, C RNA values based on the N1 assay decreased from 42,268 ± 7,608 copies/L to 14,199 ± 2,556 copies/L by the end of August (66.4% drop). As for WWTP2, a sharper decline can be observed where C RNA (N1) of WWTP2 reduced from 542,056 ± 25,775 copies/L to 13,313 ± 2,396 copies/L which represents a fall of 97.6%. Lowest C RNA (N1) of WWTP3 was registered at the end of July 2020 where it dropped 161,877 ± 29,138 copies/L to 7,880 ± 1,420 copies/L. C RNA (N1) profile in WWTP4 dropped sharply after 8 th July 2020 and Standard deviations shown in the bar charts were based on duplicate measurements.

March to August 2020. S1-9 represents the sampling days conducted between 21 st June to 30 th August 2020 These sampling days coincide with the period where the number of positive cases in Qatar was declining after reaching a peak in late May 2020 (Abu-Raddad et al., 2020a) . As the daily reported SARS-CoV-2 positive cases in Qatar decreased by over 66% between 21 st June and the end of August 2020, declining C RNA trends were also observed in all the WWTPs by the end of the study period. For the C RNA counts based on the N2 assay, overall decreasing patterns were also observed albeit with a larger fluctuation between the readings. It is interesting to note that a momentary surge in the C RNA profile can be observed in almost all the WWTPs during the first two weeks of August 2020 which coincided with the rise in the daily reported SARS-CoV-2 J o u r n a l P r e -p r o o f positive cases in Qatar (circled in Figure 3 ). This upswing was attributed due to the increase of social and family gatherings as well as reduced caution against COVID-19 transmission risks during national holidays that were declared in the first week of August 2020 in conjunction with the muted Eid Al Adha celebration (Qatar Tribune, 2020) .

The dynamic C RNA profile of each WWTP could provide useful information in managing COVID-19 spread. For instance, while all other WWTPs displayed a decreasing trend from June to the end of July 2020, C RNA profile in WWTP1 was ascending prior to decline after mid-June.

On the other hand, the C RNA profile of WWTP4 remained fairly constant after the initial decrease in mid-July 2020. These different profiles could potentially assist local authorities to deploy targeted approach and manage COVID-19 hot zones in the country by monitoring the C RNA profiles of each wastewater treatment plant. It is worth to mention that identification of hot zone based on individual WWTP might not be straightforward in Qatar as some of the WWTPs were also served by sewage tankers that operate from various locations.

The C RNA values reported in this study are in a similar range to those reported by Sherchan et al. (2020) but are three to four orders higher than the concentration reported by . Differences between studies in different regions can be attributed to many varied factors, such as the efficiency of recovery of RNA from the wastewater samples, the variability in the severity and shedding of virus from positive cases, losses in the wastewater samples, environmental conditions such as temperature, dry or wet season, and possibly other unknown parameters. (Equation 2). This calculation confirmed that these five WWTPs served the majority of the population in Qatar which is approximately 2.8 million (Planning and Statistics Authority of Qatar, 2020). The estimated total infected population calculated using the mathematical model (Table S3 ) is much higher than the daily reported positive cases, as has been the case in other studies . This is expected as the C RNA values capture both the diagnosed and undiagnosed cases, (a substantial number of population are either asymptomatic or paucisymptomatic and may not be documented (La Rosa et al., 2020) . Indeed, a populationbased survey in Qatar found that 58.5% of those PCR positive did not report any symptoms in the preceding 14 days.

A nation-wide seroprevalence study of craft and manual workers in Qatar has found that only 9.3% of those antibody-positive had a prior PCR laboratory-confirmed infection. This is still a higher diagnosis ratio than that reported for 15 other countries at 5.5 % (Phipps et al., 2020) , possibly because of the high testing rate in Qatar at 573,000 tests per million population, as of October 29, 2020, one of the highest worldwide (Abu-Raddad et al., 2020a Jeremijenko et al., 2020) . An epidemiologic mathematical modeling study of the Qatar epidemic Our results did not indicate that SARS-CoV-2 had appeared earlier in the wastewater than the confirmed virological laboratory testing as reported by others (Medema et al., 2020; Trottier et al., 2020) . Nemudryi et al. (2020) opined that the detection of SARS-CoV-2 RNA in wastewater could closely overlap with the virological laboratory test dates, with SARS-CoV-2 RNA detectable in the wastewater 5-8 days after infection. As hospitalization/clinical testing generally occurs between 3-9 days after the onset of the symptoms, there is a possibility for these events to coincide.

It was observed that the model tends to be underpredicting when the number of infected cases was lower in Qatar, when compared to the corrected 22 days of cumulative cases. During the period of this study, the number of SARS-CoV-2 cases in Qatar had passed the peak of the first wave and was in the decline phase. Losses in the RNA during in-sewer transport, sample processing and extraction are inevitable, and quantification of losses is difficult. A sizable portion of RNA can be lost in the primary sludge removed during influent pre-processing, with higher RNA concentrations found in the primary sludge than in the aqueous influent phase (Alpaslan Kocamemi et al., 2020; Westhaus et al., 2021) . Less than ideal recovery of RNA during the sample processing can also be exacerbated when lower amounts of RNA are present.

The huge variability in the viral shedding in the stool is another crucial factor that governs the accuracy of the estimation of the infected population . We applied a constant diagnosis rate throughout the sampling period, however actual diagnosis rate may have varied due to testing capacity, conducting of wide scale surveillance testing, and demographics of the population affected at any given time. Epidemiological modeling in Qatar indicated that the J o u r n a l P r e -p r o o f diagnosis rate increased steadily with time, explaining in part the differences seen in the trends of the 22-day cumulative cases and the estimated number of infections (Figure 4) .

The economic implications and practical limits of medical screening for the SARS-CoV-2 (COVID-19) have rapidly come into the spotlight on a global scale. Following promising results in early 2020, public health authorities in many countries have effectively implemented the use of wastewater-based epidemiology (WBE) as a potential tool for assessing and monitoring the spread of SARS-CoV-2. Based on the findings of this study, it can be concluded that the WBE approach is suitable for monitoring the trend in the numbers of infected people in Qatar. This approach was shown to be more reliable in identifying the true magnitude of infection circulating within the population than the RT-qPCR diagnostic testing using Bertollini of MoPH for their support. We also thank QEERI HSSE, HBKU facility teams for ensuring the work is safely conducted.

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Writing -original draft

Writing -review & editing. Andrew Jeremijenko: Resources Hussein A Abu Halaweh: Resources. Jenny Lawler: Conceptualization, Resources, Methodology, Writing -review &editing, Supervision. Khaled A Mahmoud: Conceptualization, Resources, Methodology, Writing -review &editing, Supervision, Project administration

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.J o u r n a l P r e -p r o o f