The new coronavirus (SARS-CoV-2) is a respiratory virus causing coronavirus disease (COVID-19). Individuals with COVID-19 can shed the viral genome in their feces, even if they do not have symptoms, and the virus can be detected in wastewater. The current study provides the first surveillance of SARS-CoV-2 RNA genome in the wastewater in Egypt. To study this aim, untreated influent (n = 48) and treated effluent (n = 48) samples were collected between January and December 2021 from the wastewater treatment plant in Giza. The viral RNA genome was determined by reverse transcription-polymerase chain reaction (RT-PCR) (S, E, and N target regions) and real-time quantitative reverse transcription-PCR (RT-qPCR) (N1 and N2 target regions). The RT-PCR assay failed to detect SARS-CoV-2 RNA in all samples analyzed, whereas RT-qPCR succeeded in the detection of N gene of SARS-CoV-2 in 62.5% of untreated influent samples. The RT-qPCR Ct values of those samples tested positive ranged from 19.9 to 30.1 with a mean of 23. The treated effluent samples were negative for viral RNA detected by both RT-PCR and RT-qPCR, indicating the efficiency of the sewage treatment plant in degrading SARS-CoV-2. Our preliminary findings provide evidence for the value of wastewater epidemiology approach for the surveillance of SARS-CoV-2 in the population to assist in the responses of public health to COVID-19 outbreak.
SARS-CoV-2 was detected in 62.5% of untreated influent samples.
RT-qPCR was more sensitive than RT-PCR.
The RT-qPCR Ct values ranged from 19.9 to 30.1 with a mean of 23.
There was a positive correlation between the number of sewage samples tested positive for SARS-CoV-2 RNA and the daily count of new active COVID-19 cases.
Since the 1918 influenza pandemic, SARS-CoV-2 has spread fast worldwide to become the most important global health threat (Ceylan et al. 2020), leading to 448,154,090 diagnosed cases and 6,027,317 deaths as of March 5, 2022 (Worldometer 2020; WHO 2022). Transmission of SARS-CoV-2 between people occurs mainly by large respiratory droplets, small aerosols, and fomites (WHO 2020). Moreover, more than 80% of patients with COVID-19 shed the SARS-CoV-2 genome in their stool (Foladori et al. 2020; Guo et al. 2021). The level of viral genome shed in the feces of patients with COVID-19 ranged from 103 to 107.6 genomic copies/g of fecal material (Foladori et al. 2020). This opens potential routes of fecal–oral transmission. Consequently, several studies across the world have monitored the presence of SARS-CoV-2 RNA in municipal sewage or wastewater, sewage sludge, medical wastewater, river water, and secondary-treated wastewater (Haramoto et al. 2020; Rimoldi et al. 2020; Zhang et al. 2020; Ahmed et al. 2021; Barril et al. 2021; Fongaro et al. 2021; Zhao et al. 2022).
Wastewater-based epidemiology (WBE), based on the wastewater analysis to monitor infectious disease, is an effective surveillance method to evaluate the real status of infectious disease outbreak in a certain region by investigating the viral concentration in the wastewater, as it represents viral shedding from both asymptomatic and symptomatic individuals, which may not be estimated by the clinical surveillance system (Choi et al. 2018; Xagoraraki & O'Brien 2020). In fact, some recent reports showed a clear positive link between the number of COVID-19 cases in the population and viral titers in the wastewater (Medema et al. 2020; Peccia et al. 2020). The WBE tool has been used effectively during the past enteric viral outbreak, such as poliovirus, human norovirus, and hepatitis A virus (Hellmér et al. 2014; Duintjer Tebbens et al. 2017).
Several recent studies from various countries including the Netherlands (Izquierdo-Lara et al. 2021), India (Chakraborty et al. 2021), Italy (Rimoldi et al. 2020), Germany (Westhaus et al. 2021), USA (Green et al. 2020), France (Trottier et al. 2020; Bertrand et al. 2021), Turkey (Kocamemi et al. 2020), Pakistan (Yaqub et al. 2020), Japan (Hata et al. 2021), Australia (Ahmed et al. 2020), and Spain (Balboa et al. 2020) have documented the SARS-CoV-2 RNA in both treated and untreated wastewater. Furthermore, SARS-CoV-2 was shown to be negative in the outflow of wastewater treatment plants (WWTPs) equipped with tertiary disinfection, whereas the treated sewage from secondary wastewater treatments have been shown to be positive for SARS-CoV-2 (Randazzo et al. 2020). In addition, presence of SARS-CoV-2 RNA in river water, marine sediments, bivalve mollusks, and sewage sludge was reported (Peccia et al. 2020; Fongaro et al. 2021; Polo et al. 2021). To date of submission of this publication, there is no environmental monitoring of SARS-CoV-2 in WWTPs in Egypt. Thus, in the current study, we aimed to test the presence of the SARS-CoV-2 RNA in urban wastewater from Giza governorate, Egypt.
Wastewater samples consisting of untreated influent (n = 48) and treated effluent (n = 48) were weekly collected between the months of January and December 2021 from the Zenin WWTP. Samples (500–1,000 ml) were collected by grab sampling technique and transported on ice to the laboratory and stored at 4 °C until analysis.
Virus concentration by polyethylene glycol precipitation
Viruses were concentrated from 250 ml of wastewater using a polyethylene glycol (PEG) precipitation (Borchardt et al. 2017), with some modification. Prior to viral concentration steps, samples were shaken at 4 °C at 100 rpm for 30 min to transfer viruses to the aqueous phase. Bacterial debris and large particles were removed from the samples by centrifugation at 4,600 rpm for 60 min at 4 °C (Hettich, Rotanta 460R, Tuttlingen, Germany). The supernatant was mixed with PEG 6000 (10% w/v) and 0.2 M NaCl (1.8% w/v) by shaking for 2 min. The mixture was divided in six 50-ml falcon tubes. Viruses were precipitated by centrifugation at 4,600 rpm for 150 min at 4 °C. The supernatant was removed carefully without disturbing the pellets. Pellets of each falcon tubes were re-suspended with 1 ml of phosphate buffer saline and stored at –20 °C.
Viral RNA extraction
The RNA extraction from 300 μl of concentrate was performed using QIAamp Viral RNA isolation kit (Qiagen, Hilden, Germany), as per the manufacturer's guidelines. The procedure resulted in 50 μl of extracted RNA. Nuclease-free water was used as a negative control of isolation with each viral RNA extraction set to monitor possible cross contamination. To examine polymerase chain reaction (PCR) inhibition, a representative sample was inoculated with 4.7 × 108 GC/ml murine norovirus (MNV-1) as sample process control virus (SPCV) (previously analyzed negative for MNV-1) by quantitative polymerase chain reaction (qPCR) (Lee et al. 2015), and no inhibitory effects could be observed.
cDNA was synthesized by the reverse transcription with random hexamers based on manufacturer's protocol for each reagent used. About 8 μl of RNA was mixed with 1 μl of random hexamer primer (0.2 μg/μl) and then the mixture was denatured for 5 min at 56 °C and kept immediately on ice. A mixture containing 2 μl of 10× M-MuLV buffer, 1 μl of 10 mM dNTPs, and 1 μl of M-MuLV RT was added to the reaction mixture and the final volume was made up to 20 μl with nuclease-free water. cDNA synthesis was carried out at 25 °C for 5 min, 42 °C for 60 min, and then at 65 °C for 20 min. To amplify E, S, or N gene using specific primers described by Park et al. (2020), 2 μl of cDNA was mixed with the PCR mixture containing 4 μl of 5× Phusion HF buffer, 0.4 μl of 10× mM dNTPs, 1 μl of forward primer or reverse primer (0.5 μM for each), and 0.2 μl of fusion high-fidelity polymerase, and then the reaction volume was made up to 20 μl with nuclease-free water. The PCR cycling condition was as follows: 98 °C for 30 s followed by 35 cycles at 98 °C for 10 s, 72 °C for 1 min with final extension step at 72 °C for 10 min. The amplicons were subjected to electrophoresis in a 2% agarose gel at 120 V for 30 min and visualized using ethidium bromide under UV light.
The occurrence of SARS-CoV-2 RNA in wastewater was detected by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) using the kit of artus Qiagen (Germany, Kit # 4511440), according to the manufacturer's instructions. This kit included the N1 and N2 primers and probe mix to target the nucleocapsid genes (N gene: N1 and N2) of SARS-CoV-2, which are described by the US Centers for Disease Control and Prevention design (CDC 2020). The final reaction volume of 25 μl was prepared by mixing the viral RNA solution with 7.25 μl of primer/probe mix, 6.25 μl of mastermix. RT-qPCR reactions were performed on a Rotor-Gene Real-time PCR Detection System (Qiagen, Germany), under the following thermocycling conditions: 50 °C for 10 min, 95 °C for 2 min, followed by 40 cycles of 95 °C for 5 s and 58 °C for 30 s. Samples with a cycle quantification value (Ct ≤38) were considered positive, and each sample was quantified in duplicate, for which we provided a mean value. Negative control consists of nuclease-free water, positive control consists of a double-strand DNA amplified with the SARS-CoV-2 primers/probes mix, and an internal control consists of a single-strand IVT RNA. Sampling control detects the RNAse P gene included in each assay.
|Month .||Untreated influent|
|No. of samples tested .||No. of positive samples .||Ct values .||No. of samples tested .||No. of positive samples .||Ct values .|
|Month .||Untreated influent|
|No. of samples tested .||No. of positive samples .||Ct values .||No. of samples tested .||No. of positive samples .||Ct values .|
a RT-qPCR Ct values ≥38.
bMean scores of RT-qPCR Ct values.
Based on previously published reports, SARS-CoV-2 genome has been found in raw sewage from Australia (Ahmed et al. 2020), USA (Wu et al. 2020; Sherchan et al. 2021), Italy (La Rosa et al. 2020; Castiglioni et al. 2022), India (Kumar et al. 2020), and the Netherlands (Medema et al. 2020). However, till the date of submission of this study, there is no environmental surveillance for SARS-CoV-2 RNA in the wastewater in Egypt. Therefore, this finding from Egypt is in conformity with these recently published studies. Indeed, despite the benefits of SARS-CoV-2 surveillance in wastewater for the WBE surveillance strategy, the public health risk related to water cycle is unclear because infectious viral particles in wastewater were yet to be investigated.
In the current study, we targeted three various regions of the SARS-CoV-2 RNA genome (S, N, and E) in wastewater samples by traditional RT-PCR. Unfortunately, we failed to detect the viral genes in the sample analyzed by this assay. However, La Rosa et al. (2020) succeeded by using RT-PCR to detect ORF1ab and S genes in wastewater. This may be attributed to the low sensitivity of the primers used in this study. In addition, we used here a single PCR compared to nested-PCR which succeeded in the detection of SARS-CoV-2 RNA genome in wastewater (La Rosa et al. 2020). Jalouli et al. (2015) reported that nested-PCR improved the detection rate, sensitivity, and efficiency rate of viral detection, compared to a single PCR. Furthermore, wastewater RT-qPCR was found to be a reliable and sensitive assay for the early detection of COVID-19 outbreaks (Randazzo et al. 2020).
In the current work, we also targeted N1 and N2 regions for the detection of SARS-CoV-2 in wastewater using two primer sets (CDC N1 and CDC N2) by real-time RT-PCR. The viral N gene is the most common transcript of SARS-CoV-2 RNA and thus it is widely used for environmental surveillance of SARS-CoV-2 (Mahlknecht et al. 2021; Sherchan et al. 2021). In contrast to RT-PCR, the real time RT-PCR detected the N gene of SARS-CoV-2 in 30 (62.5%) untreated influent samples. Research in other countries reported higher positive rates (ranging from 33 to 100%) for SARS-COV-2 RNA genome in wastewater (Kumar et al. 2020; Rimoldi et al. 2020; Mahlknecht et al. 2021; Saawarn & Hait 2021). However, a lower positive rate (11.6%) than our finding was reported in a study from Czech Republic (Mlejnkova et al. 2020). Indeed, several factors may have attributed to this fluctuation, including sample type and volume, sample frequency, virus concentration protocol used, and the sensitivity primers and probes used (Ahmed et al. 2020). Moreover, SARS-CoV-2 concentration in wastewater may vary depending on many factors, like the number of infected individuals in the catchment, the amount of viral RNA shedding by COVID-19 individuals, the persistence of viral RNA in wastewater, the sewer system type, dilution and mixing, and sampling type (24-h composite or grab) and time (Ahmed et al. 2020). Overall, our finding confirmed previous studies reporting that the viral N gene can be used successfully to test the occurrence of SARS-CoV-2 RNA in sewage samples in the Netherlands (Izquierdo-Lara et al. 2021), the UK Peccia et al. 2020), Italy (La Rosa et al. 2020; Castiglioni et al. 2022), Australia (Ahmed et al. 2020), USA (Sherchan et al. 2020), and Spain (Orive et al. 2020).
Randazzo et al. (2020) already reported that WWTPs based on secondary treatment like the Zenin WWTP can release SARS-CoV-2 viral RNA in their treated effluents. However, we could not detect the viral genome in treated effluent during the entire sampling period by both RT-PCR and RT-qPCR which is in agreement with recent studies from different countries (Haramoto et al. 2020; Rimoldi et al. 2020; Kumar et al. 2021). This finding demonstrates that the wastewater treatment technologies applied in the Giza governorate are efficient in the SARS-CoV-2 removal, and confirms the safety reuse of the treated effluent across that region. In contrast with other studies, SARS-CoV-2 RNA genome was detected by the RT-qPCR assay in treated effluent and even rivers (Haramoto et al. 2020; Randazzo et al. 2020; Rimoldi et al. 2020). In this study, absence of SARS-CoV-2 RNA in the treated effluent may be also due to the low volume of samples (250 ml) which is much smaller than the volumes used in previous studies (Jeddi et al. 2022; Kaya et al. 2022; Monteiro et al. 2022). Indeed, a high sampling volume is required to increases the RT-qPCR sensitivity, particularly, when viral concentration is low in the sample (Yanaç et al. 2022). However, a large volume of sample concentrate has high concentrations of chemicals or organic matters (e.g., humic acids) that can inhibit RNA extractions and subsequent RT-qPCR detection procedures (Cashdollar & Wymer 2013; Haramoto et al. 2018).
Despite our detection to viral RNA fragments in untreated influent, we could not confirm infectious SARS-CoV-2 in those wastewater samples tested positive. Recently, discharge of wastewater into the aquatic environment has been suggested as a possible transmission route for SARS-CoV-2 (Mohapatra et al. 2021). However, Giacobbo et al. (2021) documented that SARS-CoV-2 detected in raw wastewater was not infectious.
Based on the positive detection of SARS-CoV-2 RNA genome in wastewater at the WWTP, there were limited COVID-19 recorded cases. WBE can provide an effective approach as an early warning system. Recent reports demonstrated that 40.96% of COVID-19-infected persons are asymptomatic (Ma et al. 2021). WBE allows for the detection of SARS-CoV-2 cases that are still asymptomatic or pre-symptomatic in the COVID-19 progression. One of the most difficult challenges will be to make quantitative predictions about the number of cases in the community based on viral RNA concentrations measured in the sewage. Indeed, several studies have tried to determine a relationship between the increase/decrease in the number of COVID-19 patients and concentrations of SARS-CoV-2 RNA in wastewater influents (Ahmed et al. 2020; Randazzo et al. 2020; Wurtzer et al. 2020). For instance, a previous study from France documented a relationship between the increase in the number of COVID-19 cases and untreated influent viral RNA load in the region (Wurtzer et al. 2020).
A significant positive relationship was found between the number of positive sewage samples for SARS-CoV-2 RNA and the daily count of new active COVID-19 cases in the Egyptian population. The same relationship has been reported in previous studies from other countries during the pandemic of COVID-19 (Medema et al. 2020; Peccia et al. 2020; Weidhaas et al. 2021). Moreover, low RT-qPCR Ct values (ranging from 19.9 to 30.1) were observed in this study. This finding refers to the high numbers of infected persons in the monitored area. Notably, the lowest Ct values (ranging from 19.9 to 23.8) were observed in samples collected during both waves, indicating that the viral N gene was more abundant. This finding is consistent with previous studies, suggesting that WBE could be employed as an early warning tool for detection and spread of SARS-CoV-2 (Rimoldi et al. 2020; Ahmed et al. 2021; Gerrity et al. 2021). It is worth mentioning that eight positive samples with the highest Ct values were also found between the two waves and earlier than the fourth wave. The decline in viral RNA in wastewater may be due to the decline in case counts (Weidhaas et al. 2021). Moreover, Randazzo et al. (2020) confirmed that SARS-CoV-2 RNA could be detected in influent wastewater several weeks prior the initial confirmed case. Furthermore, long-term SARS-CoV-2 shedding following negative swab tests could explain the finding of viral RNA in wastewater following the drop in case counts. However, further studies would be performed to confirm these findings during the decline in case counts and outbreaks in a sewershed at multiple locations.
Nevertheless, the current work had some limitations. First, wastewater samples were stored at −20 °C for 1–2 weeks until further processing, and this may have influenced our results. However, Ahmed et al. (2020) reported the average time needed to reduce 1-log10 of SARS-CoV-2 viral RNA genome in raw wastewater ranged from 1 to 3 weeks. Therefore, the viral RNA can last long enough in aquatic environment for accurate detection. Second, the lack of sequencing data due to the little amount of DNA generated by the RT-qPCR and it cannot be used for the study's aim. This problem has also been stated in other reports (Stals et al. 2011; Felix-Valenzuela et al. 2012). Third, the RT-qPCR procedure used in this study cannot distinguish between the presence of replication-competent (i.e., infectious) SARS-CoV-2 and remnant viral traces (i.e., non-infectious) in the positive samples. However, no infectious SARS-CoV-2 particles were found in wastewater samples using cell lines (Rimoldi et al. 2020), and this may be due to the sensitivity of viral protein capsid to the organic solvents and detergents (Ye et al. 2016).
This study shows the first environmental surveillance for SARS-CoV-2 RNA in Egypt, demonstrating that SARS-CoV-2 RNA, or even their virions, may reach river waters. Moreover, the occurrence of SARS-CoV-2 in wastewater samples in Egypt demonstrates the WBE significance in active surveillance of SARS-CoV-2 at the population level, in contrast to clinical surveillance. Further studies are needed on the monitoring and persistence of SARS-CoV-2 RNA genome in different water matrices. Also, in the future, studying the correlation between the SARS-CoV-2 viral concentrations to the number of COVID-19 individuals in the monitored areas will be performed.
We would like to acknowledge the STDF-Egypt/Science and Technology Development Fund (Grant No. 43703) to Y.E.S. for the financial support to the current research. The funder had no role in study design, data collection, data analysis, or preparation of the manuscript and decision to publish.
DATA AVAILABILITY STATEMENT
All relevant data are included in the paper or its Supplementary Information.
CONFLICT OF INTEREST
The authors declare there is no conflict.