key: cord-333547-88dkh6xd
authors: Hasan, Shadi W.; Ibrahim, Yazan; Daou, Marianne; Kannout, Hussein; Jan, Nila; Lopes, Alvaro; Alsafar, Habiba; Yousef, Ahmed F.
title: Detection and Quantification of SARS-CoV-2 RNA in Wastewater and Treated Effluents: Surveillance of COVID-19 Epidemic in the United Arab Emirates
date: 2020-10-19
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2020.142929
sha: 
doc_id: 333547
cord_uid: 88dkh6xd

Testing SARS-CoV-2 viral loads in wastewater has recently emerged as a method of tracking the prevalence of the virus and an early-warning tool for predicting outbreaks in the future. This study reports SARS-CoV-2 viral load in wastewater influents and treated effluents of 11 wastewater treatment plants (WWTPs), as well as untreated wastewater from 38 various locations, in the United Arab Emirates (UAE) in May and June 2020. Composite samples collected over twenty-four hours were thermally deactivated for safety, followed by viral concentration using ultrafiltration, RNA extraction using commercially available kits, and viral quantification using RT-qPCR. Furthermore, estimates of the prevalence of SARS-CoV-2 infection in different regions were simulated using Monte Carlo. Results showed that the viral load in wastewater influents from these WWTPs ranged from 7.50E+02 to over 3.40E+04 viral gene copies/L with some plants having no detectable viral RNA by RT-qPCR. The virus was also detected in 85% of untreated wastewater samples taken from different locations across the country, with viral loads in positive samples ranging between 2.86E+02 and over 2.90E+04 gene copies/L. It was also observed that the precautionary measures implemented by the UAE government correlated with a drop in the measured viral load in wastewater samples, which were in line with the reduction of COVID-19 cases reported in the population. Importantly, none of the 11 WWTPs’ effluents tested positive during the entire sampling period, indicating that the treatment technologies used in the UAE are efficient in degrading SARS-CoV-2, and confirming the safety of treated re-used water in the country. SARS-CoV-2 wastewater testing has the potential to aid in monitoring or predicting an outbreak location and can shed light on the extent viral spread at the community level.

Coronaviruses (CoVs), which range from 60 to 220 nm in size, are enveloped positive-sense single-stranded ribonucleic acid (RNA) viruses with club-like spikes on their surface, and infect a wide range of hosts including birds and mammals. They were first identified in the mid-1960s, and have traditionally been associated with the common cold in humans. More recently, they gained significant medical importance due to the emergence of three deadly zoonotic strains: (i) severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 (mortality rate 11%), (ii) Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 (mortality rate 35%), and (iii) SARS-CoV-2 in 2019 (mortality rate still being determined, but estimated at 2-3%) [1] [2] [3] [4] [5] .

CoVs have more detrimental impacts on immunocompromised and elderly individuals and have the potential to create worldwide outbreaks [6] . The first cases of SARS-CoV-2 infections occurred in either late November or early December 2019, and quickly spread until the World Health Organization (WHO) declared it a Public Health Emergency of International Concern on January 30 th 2020, and a pandemic on March 11 th 2020. Despite international efforts to contain the virus using various policies, it is still posing a serious global challenge today [7, 8] . Scientists attribute the difficulty in managing the outbreak to a long incubation period (~5 days) and viral shedding and transmission by asymptomatic infected individuals [9, 10] .

Most of what we know about viral shedding comes from studies that quantify SARS-CoV-2 in pharyngeal or nasopharyngeal swabs, with very little work done to understand the shedding of this virus in stool. A limited number of studies have shown that the shedding period of SARS-CoV-2 in stool samples varies considerably, and can still be detected up to 27.9 ± 10.7 days after infection in some cases [9, 11] . The major transmission routes of SARS-CoV-2 have been shown to be through aerosol droplets, person to person contact, and contaminated surfaces J o u r n a l P r e -p r o o f Journal Pre-proof [11] [12] [13] [14] [15] . Some studies demonstrated that the virus can replicate in cells that line the human digestive tract, and that a significant number of COVID-19 patients have detectable levels of viral RNA in their stool [16] [17] [18] . In some cases, the number of viral gene copies in one gram of stool ranges from ~10 4 to 10 8 genomes [16] [17] [18] . Even though the virus is detectable in the stool of COVID-19 patients, there is limited evidence that it is infectious, as only one study to date has demonstrated this to be the case, while another study demonstrated that it was not possible to isolate infectious viruses from the stool of COVID-19 patients [16] [17] [18] . These findings indicate that SARS-CoV-2 could be detected in the municipal wastewater of a region containing infected individuals. Furthermore, it also means that monitoring the virus in municipal wastewater represents an effective low-cost method to track infections in the population.

Wastewater monitoring has been a successful method of tracking emerging contaminants and circulating antimicrobial/antibiotic resistance genes [19] [20] [21] . Furthermore, monitoring different types of viruses, such as Hepatitis A, Noroviruses, and Rotavirus, in wastewater has also been used as a surveillance technique to investigate the distribution of these viruses in a community, a practice termed water-based epidemiology (WBE) [22, 23] . It is thus possible that WBE and the environmental surveillance of SARS-CoV-2 in wastewater could help track the outbreak in a certain region and identify regions with infected asymptomatic individuals.

Carrying out SARS-CoV-2 testing to identify and quarantine affected individuals is a good strategy for managing this outbreak in countries with testing capabilities and capacities, and is the method used here in the UAE. However, it is a reactionary strategy that can only be deployed after a case is reported clinically, and by then it is likely that the virus has been spreading through a population at a significant level. In contrast, detecting and quantifying SARS-CoV-2 in wastewater covers a larger portion of the population as it essentially tests J o u r n a l P r e -p r o o f Journal Pre-proof everyone who contributed to the wastewater sample analyzed [24, 25] . If successful, it would be a way of mass testing and could lead to early detection before infections manifest as clinical cases or hospital admissions. Recent peer-reviewed and non-peer-reviewed studies published by several groups in the Netherlands, the USA, France, Spain, Italy, Australia, and Turkey have demonstrated that the idea of tracking infection rates by measuring the viral load in wastewater is feasible, cost-effective, and efficient [1, [26] [27] [28] [29] . The authors believe that the viral concentration in wastewater is a good predictor of the number of infected individuals in the population of the United Arab Emirates (UAE), including asymptomatic cases that would otherwise remain undetected. Most importantly, monitoring wastewater could be used as a way of tracking the prevalence of the virus and become an early-warning tool for new outbreaks in the future.

Consequently, the main objectives of this study were: (i) to detect the presence of SARS-CoV-2 virus in municipal (untreated) wastewater and treated effluents of wastewater treatment plants (WWTPs) in the UAE; (ii) to quantify the viral concentration in viral gene copies per liter; and (iii) to explore whether these measurements mirror infections in the population in order to comment on the utility of this method to track the epidemiology of the disease. Thus far, the frequency and degree of testing individuals and wastewater samples achieved in the UAE have been replicated only in a few other jurisdictions. To the best of our knowledge, this is the first study to detect and quantify SARS-CoV-2 in wastewater in the region. various sewer access points (e.g. manholes located in neighborhoods) and pumping stations.

These 11 WWTPs implement a series of treatment technologies including preliminary, primary, secondary (ASP/clarification), and tertiary (sand filtration, disinfection, chlorination) for the purpose of reusing treated water (TSE) for irrigating farmland and watering green spaces.

Composite samples were collected over a 24-hour period using auto-samplers, and 250 mL of the composite samples were transferred to ISOLAB GmbH sterile polypropylene (PP) plastic bottles, Labortechnik GmbH, Germany) for 90 min [30] [31] [32] . Although this inactivation step might lead to some viral loss, it is an essential safety measure that minimizes the risk of SARS-CoV-2 transmission to laboratory personnel. Studies have demonstrated that thermal deactivation at lower temperatures, similar to the one chosen in this study, resulted in a non-infectious virus that could still be detected by RT-PCR based methods, while deactivation at much higher temperatures (92 o C) resulted in complete degradation of the virus so that detection was not possible by RT-PCR [32, 33] . After heat inactivation, the samples cooled down to 4 o C prior to filtration and viral concentration. In the second method, 8,000 average molecular weight polyethylene glycol (PEG) was used to concentrate the viruses [30] . Briefly, 50 mL of the filtrate was transferred to a 50 mL conical tube, followed by the addition of PEG and NaCl to a final concentration of 8 and 1.8%, respectively. The sample was mixed on a nutator for 1 h in order to dissolve the PEG and NaCl and precipitate viruses. The sample was then centrifuged at 12,000 x g using a fixed angle rotor for 2 h at 4 o C. The supernatant was poured off and the pellet was re-suspended in 1.5 mL of TRIzol (ThermoFisher Cat#15596). Isopropanol (1 mL) was added to the aqueous layer and the mixture was agitated by inverting the tube before incubation at room temperature for 10 min. The RNA was pelleted by centrifugation at 12,000 x g for 15 min at 4 o C and washed once in 75% ethanol prior to resuspension in elution buffer (40 µL) from the ABIOpure Viral DNA/RNA Extraction kit. The two protocols described for viral concentration and RNA extraction (either ultrafiltration columns/RNA extraction kit or PEG/TRIzol) were selected and tested because they were the two methods that were reported for this type of work at the time these experiments were initiated in early April 2020 [30, 34] . The authors wanted to compare them to decide which methodology to adopt as wastewater sampling continued. All the data reported in the tables were obtained using the ultrafiltration method ( Fig. 1a)

The extracted RNA (8 µL 

The prevalence of SARS-CoV-2 infection in a certain region was estimated using the measured viral loads in wastewater, the wastewater flow rate, the viral load in the stool of infected individuals, and the estimated daily production of stool per capita according to Eq. 2 [27] : The stool viral load (R F ) in log 10 was simulated as a log-normal distribution with a mean of 7.18 and STDEV of 0.67 [16] . Daily stool production per capita (F) (g/capita.day) was simulated as a log-normal distribution with a mean of 149 and STDEV of 95 as per the statistics reported for the high-income countries [35] . The percentage of COVID-19 patients who shed virus in their stool (ɛ) was modeled as a uniform distribution from 0.29 to 0.55, as reported by the limited number of studies on this topic thus far [11, 15, 36] . The reported data from the simulations were based on 50,000 calculations.

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

At the time when the authors initiated this study, only two studies were reported in non-peerreviewed pre-prints; one study utilized ultra-concentration columns for viral concentration followed by RNA kit extraction, while the other study used PEG to concentrate viruses followed by TRIzol extraction [30, 37] . Therefore, in order to assess the differences between these two methods and determine which one to use for our study, viruses from the same wastewater samples (50 mL) were concentrated using either PEG followed by RNA extraction with TRIzol or using a 30 kDa MWCO column followed by extraction using a viral RNA kit as described in the materials and methods. Quantification of the viral load was then performed identically on the RNA extracted from each method using RT-qPCR.

Different methods for wastewater virus concentration have been adopted by different groups around the world [27, 30, 37] . The methods fall into three major categories and involve concentrating viruses using either (i) precipitation using chemicals such as PEG or Al(OH) 3, followed by centrifugation [1, 30, 38, 39] , (ii) ultrafiltration columns with very low molecular weight cut-offs [27, 29, 34] , or (iii) binding of viruses to electronegative membranes [27, 40] .

Using ultrafiltration columns in combination with the commercial RNA kits resulted in higher counts compared to using PEG and TRIzol (Fig. 2) . Indeed, a reading of 31.7 gene copies/mL was determined using the ultrafiltration method compared to a reading of 2.6 gene copies/mL for the same sample using the PEG/TRIzol method. Consequently, all subsequent virus measurements in the wastewater samples were performed using the ultrafiltration and commercial RNA extraction kit method.

The PEG protocol tested here was used in the state of Massachusetts in late March 2020, with a significantly higher number of reported cases than what was reported in the UAE when J o u r n a l P r e -p r o o f Journal Pre-proof the measurements reported here were performed. In the Massachusetts study, high counts ranging between 50 and 300 gene copies/mL were reported in the wastewater samples [30] .

Precipitation-based methods that rely on chemicals such as PEG to concentrate viruses have a higher throughput since they are cheap and user-friendly compared to the expensive ultrafiltration column method that sometimes requires multiple rounds of centrifugation to concentrate the wastewater sample. However, using ultrafiltration columns in combination with commercial RNA extraction kits might represent a more sensitive approach, more suited to detecting lower concentrations of the virus in wastewater. The ultrafiltration method might be more appropriate for long term surveillance as well after the virus concentration drops below the detectable limit of the assay. Once that occurs, a more sensitive assay would be able to detect Both methods used have advantages and caveats [41] . For example, one drawback of concentrating viruses using the ultrafiltration method is related to the fact that it is often difficult to concentrate all viruses from the sample, as some viruses are adsorbed to the solid phase in the wastewater sample and are thus pre-filtered when the sample is passed through the 0.2micrometer filter. As more groups report the experimental protocols they used to make SARS-CoV-2 wastewater measurements, scientists need to come together to agree on standardizing the method used to perform this analysis. Whatever the method is chosen, there will always be a viral loss that results from different steps in the process. It is the author"s opinion that the most important property of wastewater monitoring is sensitivity, reproducibility, precision, and not J o u r n a l P r e -p r o o f necessarily accuracy. At this point, the value of these measurements is to observe the change over time in order to gain insight into infection rates and kinetics in the population. Laboratories with access to BSL-3 labs will lead the way in determining which methods are best suited for this type of analysis in the future.

The viral loads in the 11 WWTPs and the 38 locations in the UAE are summarized in Fig. 3 and Table 2 J o u r n a l P r e -p r o o f preceded by an elevation of SARS-CoV-2 concentration in wastewaters [43] . Indeed, several studies have explored the detection and quantification of SARS-CoV-2 in wastewater influents [27, 30, 34, 39, [44] [45] [46] . However, only a few studies have attempted to establish a correlation between the increase/decrease in the viral load in the wastewater influents and the number of infected patients [1, 27, [43] [44] [45] . 2020, 346 new COVID-19 cases were reported in the UAE, which is less than those reported on the same day of the preceding month [42] . In another study carried out in the Murcia Region (Spain), the epidemiological data on COVID-19 revealed a tight correlation between the virus prevalence (cases per 1.00E+05 inhabitants) and the viral load in the WWTP"s influents. Their study also confirmed that SARS-CoV-2 could be detected weeks before the initial confirmed case [1] .

These studies, together with our findings, provide strong evidence for the significance of environmental epidemiological surveillance in monitoring SARS-CoV-2 outbreaks, and for this surveillance method to be used as an early warning tool for possible outbreaks or "second waves". This tool can help decision-makers in developing appropriate policies to manage outbreaks in a certain area. It can also help evaluate the effectiveness of these policies in reducing the widespread transmission of the virus. Although mass testing of inhabitants for SARS-CoV-2 is an excellent option for assessing the virus prevalence, wastewater surveillance of SARS-CoV-2 viral loads can also be a good indicator of the circumstances on the ground and could direct the mass testing efforts to specific geographic locations.

In epidemiology, the reproduction number is the number of individuals an infected person interacts with and infects. This number can be significantly reduced through infection mitigating policies that a jurisdiction implements [47, 48] . The first COVID-19 case in the UAE was reported on January 29 th 2020, and that person later recovered on February 9 th 2020. The J o u r n a l P r e -p r o o f

Several months after the virus outbreak, many countries still cannot carry out enough SARS-CoV-2 tests to identify and isolate infected individuals in order to curb the epidemic. What makes the situation even more complex is that a significant amount of viral transmission is attributed to asymptomatic infected individuals or individuals with very mild symptoms [51] [52] [53] .

This also means that the number of actual infections is underestimated. Thus, augmenting testing strategies using wastewater epidemiology data represents a promising new tool in estimating prevalence in a given location.

Different conclusions can be drawn from the wastewater analyses depending on how the data is processed, essentially achieving different levels of insight into the outbreak. At the first level, viral concentration in the wastewater (gene copies/L) is not very helpful on its own, though it can give you a good overview of how an outbreak is progressing from sampling the same area over time. This assumes that the flow of water through the area does not change much between sampling dates and viral load can inform on whether the outbreak is getting better or worse.

Importantly, different areas cannot be compared to each other using this first metric only.

For instance, the viral load in viral gene copies/L for B4 WWTP gives an indication that the region served by this WWTP is significantly more affected than the region served by B2 WWTP, attempted to provide an estimate in different regions using the Monte Carlo approach and the results were reported with 95% CI based on the simulated variables. As more groups around the world report results and communicate laboratory protocols used to make these measurements, a comprehensive comparison needs to be carried out in order for scientists to agree on standardized methods. Overall, this study showed the potential of detecting SARS-CoV-2 in wastewater as an early warning and prediction tool for the spread of the disease. This monitoring can help authorities take appropriate and prompt actions to contain any potential outbreak of COVID-19

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The authors are thankful to the Ministry of Interior (MOI) in Abu Dhabi (UAE) for their financial support (Award No. CPRA-2020-027), their immense efforts in coordination and
