Detection of SARS-CoV-2 in the sewerage system in Tunisia: a promising tool to confront COVID-19 pandemic

Aim: The current study undertaken in Tunisia examines the use of wastewaters to monitor SARS-CoV-2 circulation. Materials & methods: Viral genetic materials collected in wastewaters during two different periods (September–October 2020 and February–April 2021) were concentrated using the adsorption-elution method. SARS-CoV-2 genes were researched by real-time PCR. Results: During the first period of the study, viral RNA was detected in 61.11% of the analyzed samples collected from Monastir city with a rate of 88.88% for raw wastewaters and 33.33% for treated wastewaters. Then, during the second period of the study, the quantitative analysis of wastewaters collected from seven governorates showed the presence of viral RNA among around 25% of them with variable RNA loads. The increased amounts of viral RNA detected in wastewaters were accompanied by an increase in the number of COVID-19 patients in Tunisia. Conclusion: Our results emphasize the importance of sewage survey in SARS-CoV-2 tracking.


Aim:
The current study undertaken in Tunisia examines the use of wastewaters to monitor SARS-CoV-2 circulation. Materials & methods: Viral genetic materials collected in wastewaters during two different periods (September-October 2020 and February-April 2021) were concentrated using the adsorptionelution method. SARS-CoV-2 genes were researched by real-time PCR. Results: During the first period of the study, viral RNA was detected in 61.11% of the analyzed samples collected from Monastir city with a rate of 88.88% for raw wastewaters and 33.33% for treated wastewaters. Then, during the second period of the study, the quantitative analysis of wastewaters collected from seven governorates showed the presence of viral RNA among around 25% of them with variable RNA loads. The increased amounts of viral RNA detected in wastewaters were accompanied by an increase in the number of COVID-19 patients in Tunisia. Conclusion: Our results emphasize the importance of sewage survey in SARS-CoV-2 tracking. Afterward, during the second period of our study, we have broadened the extent of our research to cover seven regions throughout the country (Figure 1). We received 31 raw wastewater samples during the period from 24 February 2021 to 4 May 2021 (Table 2).

Viral genetic materials concentration
Viral genetic materials were concentrated by the adsorption-elution method using aluminum hydroxide and beef extract as described by [9] with minor modifications. The raw wastewater samples (2 L) were subjected to a coarse filtration, this step was omitted for treated wastewaters (effluents). Then, the filtered wastewaters were re-filtered through 0.45 μm membranes. Then, these membranes were cut and placed in 250 ml polyropylene copolymer (PPCO) centrifuge bottles. An amount of 100 ml of the obtained filtrate were added to the membrane pieces and vigorously vortexed to detach the viral particles stuck to the membranes. PPCO bottles were centrifuged at 2000 rpm for 5 min and supernatants were collected. Afterward, 100 ml of collected supernatants were placed in a PPCO centrifuge bottle, pH was adjusted to 6.0 and aluminum hydroxide solution (0.9 N) was added to the wastewaters sample (1:100). The pH was readjusted to 6.0 and the sample was shaken using an orbital shaker at 150 rpm for 15 min at room temperature to allow virus absorption, and precipitates were recovered by centrifugation at 1700 g for 20 min. Then, viral genetic materials elution was carried out by resuspending the obtained pellet in 10 ml of 3% beef extract (pH 7.4). Viral suspension was then transferred in 50 ml PPCO centrifuge tubes and shaken for 10 min at 150 rpm Afterward, viral genetic materials were recovered by centrifugation at 1900 g for 30 min and the pellet was re-suspended in 1 ml of phosphate-buffered saline. The obtained concentrates were aliquoted and conserved at -80 • C until being used.

Viral RNA extraction
Before proceeding to RNA extraction, viral concentrates were spiked with 10 μl of exogenous viral RNA which is composed of MS2 bacteriophage genome (provided in the kit used in real-time PCR experiments). This internal control material enables to verify the efficiency of RNA extraction, reverse transcription, PCR steps to demonstrate proper specimen processing, and the absence of amplification inhibitors. RNA was extracted from 300 μl of concentrates using the RNeasy PowerWater Kit (Qiagen) according to the manufacturer's instructions. The RNA was eluted in 50 μl of RNase-free water, aliquoted and conserved at -80 • C until being used for viral RNA detection.
Qualitative & quantitative detection SARS-CoV-2 RNA During the first period of the current study, qualitative analysis for the presence of SARS-CoV-2 RNA in wastewaters was performed using the Allplex 2019-nCoV kit (Seegene, Seoul, South Korea) allowing the detection of three viral targets: the E, N and RdRp genes by real-time reverse transcriptase polymerase chain reaction (RT-PCR).
Manufacturer's instructions have been followed in preparation for real-time PCR and thermal cycling conditions.  Reaction mix (25 μ) consisted of 5 μl of 2019-nCoV MOM containing primers and probes, 5 μl of 5X real-time One-step buffer, 5 μl of RNase-free water, and 2 μl of real-time one-enzymes. The thermal cycling conditions were as RT at 50 • C for 20 min, preheating at 95 • C for 15 min, and 45 cycles of amplification at 94 • C for 15 s and 58 • C for 30 s. Each sample was analyzed in triplicate and every real time RT-PCR assay included negative (RNase-free water) and positive controls (SARS-CoV-2 RNA provided in the kit). In addition, the positive signal for the internal control indicates that all steps performed from RNA extraction to the amplification of viral RNA were successful. The threshold cycle was set to 40 for all target genes and samples which were found positive for at least the N or the RdRp genes were considered positive as recommended by the manufacturer. In case of positivity for only the E gene, the sample is considered uncertain.
During the second period of our study, we performed a quantitative analysis for SARS-CoV-2 RNA using the QuantiTect virus Kit (Qiagen, Hilden, Germany) enabling a one-step quantitative detection of the viral RNA targets. We targeted the gene N of SARS-CoV-2, two fragments of this latter were amplified namely N1 and N2. CDC Primers/probes were used in the RT-PCR reaction at the concentration recommended by the CDC. Each sample was analyzed in triplicate and every real time RT-PCR assay included negative (RNase-free water) and positive controls (SARS-CoV-2 RNA, Qiagen). The threshold value was set to 0.03 and the cycle threshold was set to 40. The standard curve was constructed using single stranded RNA fragments of SARS-CoV-2 containing the target region: gene N (Joint Research Centre, EURM-019).

SARS-CoV-2 RNA detection in raw & treated wastewaters
During the first period of our study between 8 September 2020 and 2 October 2020, nine influent and nine effluent wastewater samples were investigated for the presence of SARS-CoV-2 RNA. Samples were considered positive for Ct below 40 for at least one of the two targeted genes N and RdRp as done in the previous studies in which the threshold cycle for positivity was set to 40. Out of the 18 samples analyzed, 11 were positive for viral RNA (61.11%) ( Table 1). Among the 11 positive samples, ten (90.90%) were positive for the gene N, six (54.54%) were positive for the gene E and ten (90.90 %) were positive for the gene RdRp. The highest number of positive samples was recorded in samples collected from WWTP of Frina (Table 1). 88.88% of tested raw wastewater samples (eight samples out of nine) were positive for at least one of the targeted genes (Table 1). SARS-CoV-2 RNA was also detected in three of effluent wastewater samples (n = 9) namely in Sahline and Frina WWTPs (Table 1) following a secondary treatment with activated sludge.
During the second period of our study, we have tested 31 wastewater samples which were collected from 14 different districts throughout the country. 25.80% of the analyzed samples contained SARS-CoV-2 RNA with a quantities ranging from 0.18 10 3 copies/100 ml to 59,94 10 3 copies/100 ml of wastewaters.
During the first period of our study, the detection of SARS-CoV-2 genetic materials in wastewaters has been combined with a rapid increase in the number of COVID-19 cases in the studied region (region of Monastir). For example, the cumulative number of reported COVID-19 cases in Monastir went from 104 cases in 8 September 2020 to 1288 cases in 2 October 2020 (data published by the Tunisian Ministry of Public Health). In parallel, the concentration of SARS-CoV-2 RNA in the sewage of each of the municipalities increased as indicated by a decrease in Ct values. The results obtained align with the pandemic surge recorded in Monastir city during the study period ( Figure 2).
Concerning the other regions included in the second period of our study (February-May), clinical data, such as the number of COVID-19 cases, were not available and therefore a comparison between the number of COVID-19 cases and the concentration of SARS-CoV-2 RNA in the sewage was not possible.

Discussion
Our results are in accordance with recent studies undertaken around the world demonstrating the presence of SARS-CoV-2 RNA in raw wastewater samples [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. The number of studies that have been interested in detecting the traces (RNA) of the novel coronavirus in the wastewaters has continuously increased since the emergence of the SARS-CoV-2 pandemic. This indicates the importance of sewage surveillance as a sensitive tool for viral circulation monitoring in a population, in order to predict the appearance of epidemics and bolster the efforts deployed in the clinical epidemiology setting. Consistence between SARS-CoV-2 RNA amounts and the number of confirmed cases was observed in previous reports dealing with WBE use to monitor SARS-CoV-2 circulation in Netherlands, Australia, France, Italy, Spain, India and Japan [7][8][9]12,15,16,24] showing that virus monitoring in sewage is a promising tool for the surveillance of COVID-19 spread in a community. Furthermore, viral genetic materials detected are predominantly shed by asymptomatic, presymptomatic, and pauci-symptomatic carriers of COVID-19 who represent around 80% of COVID-19 infections [25]. Thus, wastewaters surveillance could be used as a tool to determine the true scale of the virus spread and thereby alert to the high presence of the virus. The number of identified cases of COVID-19 is actually the visible part of the iceberg and does not reflect the true magnitude of the virus widespread within a population (reviewed in [26]). This seems to be also valid for our study, the real number of infected people in the region of Monastir at the beginning of October 2020 (1258 cases on 2 October 2020) was actually much higher as a large portion of the population was actually asymptomatic and pauci-symptomatic that contributed to a viral transmission. This may explain the fact that few days later, the number of COVID-19 has tremendously increased and a total of 2752 cases have been recorded on 11 October 2020 (Regional Direction of Health) pushing the authorities to decree partial lockdown, ban gatherings, and strengthen health measures in Monastir region. Regarding the other regions included during the second period of our study, we were unable to obtain the number of new COVID-19 cases, hospitalizations etc.
In this context, some studies have tried to address this question by developing mathematical models based on wastewater epidemiology and that try to determine the true scale of virus circulation while taking into account asymptomatic patients [7,8]. Importantly, in our study the detection of high amounts of SARS-CoV-2 RNA in wastewaters had foreshadowed the upsurge of the epidemic which highlights the potential benefits of using wastewater surveillance as an early warning system. Our results were continuously communicated to the Ministry of Health to help in making decisions aiming to contain the COVID-19 pandemic.
Viral RNA was also present in three secondary treated wastewaters. Our findings are consistent with several previous studies reporting the presence of SARS-CoV-2 in secondary treated wastewaters [8,13,14,19,27,28]. However, other studies have reported the absence of SARS-CoV-2 RNA following a secondary treatment in WWTPs [15,18,[29][30][31]. This discrepancy may be attributed to the difference in methods used in viral concentration and viral RNA detection among studies and/or methodologies used during the secondary treatment itself and its efficiency. Hence, a standardized method for wastewaters sampling, coronavirus concentration and detection should be used to be able to compare between studies and converge research activities in this thematic. Nevertheless, more effective methods in SARS-CoV-2 elimination from wastewaters are reported. These methods involve secondary treatments (Moving Bed Biofilm Reactor and Sequencing Batch Reactor), tertiary and advanced disinfection strategies (chlorination, ozonation, photo catalysis, advanced oxidation processes, filtration), and inactivation by heat and radiation (reviewed in [32]). Furthermore, novel, innovative, and ecological methods are also suggested to cope with the risk of SARS-CoV-2 transmission via wastewaters and sewage sludge reused in agriculture. For example, an interesting study undertaken by Ducoli et al. in Italy [33] recommends the incineration of sewage sludge which enables the destruction of organic micro pollutants and pathogens eventually present in the waste, but most importantly resulted ash was used as building material instead of being landfilled which brings together safety and usefulness of wastes.

Conclusion
Our study, the first of its kind in Tunisia, is an addition to a growing body of studies undertaken around the world praising and recommending the use of wastewaters to monitor SARS-CoV-2 circulation and anticipate epidemic spread. In the current study, SARS-CoV-2 RNA was detected in raw and treated wastewaters collected from different municipalities in Tunisia. This was accompanied by an increase in the number of COVID-19 cases recorded in our country which emphasize the importance of sewage survey in SARS-CoV-2 spread tracking and anticipation.

Future perspective
Unfortunately, we are still facing the COVID-19 pandemic. The virus is unceasingly mutating leading to the emergence of a new variants with increased transmissibility and pathogenicity. Hence, in the future we will attempt to monitor the presence of the new variants of SARS-CoV-2 in wastewaters which will provide an insight into determining high-risk zones and mitigating COVID-19 pandemic in Tunisia.

Summary points
• Wastewater-based epidemiology can be an efficient tool for virus circulation monitoring.
• SARS-CoV-2 RNA was detected in the wastewater treatment plants of different cities in Tunisia.
• SARS-CoV-2 RNA was detected in raw and secondary treated wastewaters.
• The research of SARS-CoV-2 genetic materials in wastewaters has contributed to virus tracking and has helped in making decisions aiming to reduce virus spread.