Tracking SARS-CoV-2 and its variants in wastewater in Tunisia

Since the end of 2019, the coronavirus disease-19 (COVID-19) pandemic has continued to affect humans and is still a serious illness with a variety of clinical manifestations and outcomes. The etiological agent of COVID-19 is SARS-CoV-2, a single-stranded positive RNA virus from the Coronaviridae family (Silva et al. 2022).

Several extremely infectious variants of concern (VOC) have emerged as a result of mutations in the SARS-CoV-2 genome (Joshi et al. 2022). VOCs are important in terms of viral pathogenicity, virulence, and transmission. In addition to being more transmissible, which might cause a more severe disease, they are also known to have less sensitivity to antibody neutralization (Davies et al. 2020; Wang et al. 2020; Joshi et al. 2022). The World Health Organization (WHO) has declared five VOCs until the end of 2022, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) (WHO 2022; Chua et al. 2023). By the end of 2020, the Delta variant was discovered for the first time in India (Singh et al. 2021) and quickly spread around the world. At the end of November 2021, Omicron virus strains were first identified in South Africa and ascribed to three sister lineages known as BA.1, BA.2, and BA.3. Two more Omicron lineages (BA.4 and BA.5) were discovered later, in early April 2022. According to the estimation of Tegally et al. (2022), the origins of BA.4 and BA.5 were in early January 2022 and mid-December 2021, respectively. The WHO classified BA.4, BA.5, BA.2.12.1, and BA.2.75 as VOC subvariants under monitoring. As of 12 May 2022, BA.4 and BA.5 were classified as VOC by the European Centre for Disease Prevention and Control (Islam et al. 2022).

Genomic surveillance of wastewater may be a powerful method for identifying, detecting, and predicting VOC in circulation to promote public health measures through an early warning system (Joshi et al. 2022). The WHO confirmed in August 2020 that wastewater influent and sewage sludge from various cities throughout the world contained SARS-CoV-2 virus RNA (Viveros et al. 2022). In more than 60 countries with over 3,000 sites, wastewater-based surveillance is a crucial part of environmental surveillance which provides near real-time data on community exposure to COVID-19 and is considered a cost-effective complement to clinical surveillance (Aguiar-Oliveira et al. 2020; Lamba et al. 2023; Naughton et al. 2023). The detection of SARS-CoV-2 RNA from wastewater has opened up the possibility of tracking circulating SARS-CoV-2 variants (Rios et al. 2021; Brunner et al. 2022; Smyth et al. 2022).

The first cases of COVID-19 positive patients in Tunisia were reported in early March 2020, and shortly thereafter, the country experienced the first COVID-19 wave. There were about 950 confirmed cases as of 26 April 2020, with 40 fatalities and a mortality rate of about 4% (Dhaouadi et al. 2023). The national authorities imposed a state of lockdown and closed the borders, which limited the disease's spread throughout the nation. The prevalence of COVID-19 declined from May to June 2020, reaching null between 4 June and 12 June 2020 (WHO 2023). The second wave began in late July 2020 and hit its peak in January 2021 (WHO 2023). Tunisia experienced the third, fourth, and fifth waves of COVID-19 after the introduction of Alpha, Delta, and Omicron variants in March, May, and December 2021, respectively (Chouikha et al. 2022). RT-PCR was implemented initially by the National Influenza Center (NIC), located at Charles Nicolle Hospital of Tunis, and then by many other public health laboratories throughout the country (Abid et al. 2020). The first SARS-CoV-2 whole-genome sequences determined from clinical samples in Tunisia were reported in June 2020 (Handrick et al. 2020). Later several other sequences were detected. Nevertheless, tracking SARS-CoV-2 and its variants in Tunisia is mainly based on clinical samples.

In the present study, we aimed to estimate the levels of SARS-CoV-2 circulation in Tunisia between December 2021 and July 2022 and detect the VOC through the monitoring of wastewater collected from six wastewater treatment plants (WWTPs) in Tunisia. Furthermore, this work is intended to report the correlations between SARS-Cov-2 wastewater concentrations and COVID-19 cases and get insights into the efficacy of wastewater treatment procedures used in the WWTP studied.

24-h composite wastewater samples (n = 44) were collected from six WWTPs in Tunisia: Sidi Bouzid (hereafter referred to as WWTP1), Monastir (WWTP2), Gabes (WWTP3), Kairouan (WWTP4), Tunis (WWTP5), and Beja (WWTP6) between December 2021 and July 2022. Sampling of untreated wastewater was carried out monthly from December 2021 to April 2022. Between May and July 2022, wastewater influent and effluent samples were collected every 2 weeks. Samples were collected in glass containers kept at 4 °C upon arrival and concentrated within 24 h.

The sample (50 ml) was passed through an electronegative membrane 0.45-μm-pore-size 47 mm diameter (Sartorius) after the addition of MgCl₂ to the sample to obtain a final concentration of 25 mM MgCl₂. Immediately after filtration, filters were stored in a −80 °C freezer until nucleic extraction. Filters were extracted within 24 h as previously described (Othman et al. 2023).

Viral RNA was extracted using the RNeasy PowerWater Kit (Qiagen) according to the manufacturer's instructions, with a minor modification. The RNA was eluted in 50 μL of RNase free water aliquoted and conserved at −80 °C until used for viral RNA detection.

The sdPCR assay was performed following the manufacturer's instructions (Qiagen, Germany) as previously described (Othman et al. 2023) using the QIAcuity one, 5plex, the Qiacuity one-step viral RT-PCR Kit (Qiagen) and SARS-CoV-2 N1 + N2 assay kit (Qiagen); for detection and quantification of SARS-CoV-2 (N1 and N2, published by CDC) in wastewater samples. Briefly, 40 μL of reaction mix was prepared for each reaction composed of 10 μL of master mix, 800 nM forward primer, 800 nM reverse primer, 250 nM probe, 0.4 μL of 100× multiplex reverse transcription mix, 7.6 μL RNase free water, and 20 μL of template RNA.

Following assembly in the pre-plate, reaction mixtures were transferred into QIAcuity Nanoplate 26k 24-well and loaded onto the QIAcuity one, 5plex. The RT-sdPCR workflow includes (i) a priming and rolling step to fill and then seal the reaction chamber partitions; (ii) an amplification step following this cycling protocol: 40 min at 50 °C for reverse transcription, 2 min at 95 °C for enzyme activation, 5 s at 95 °C for denaturation and 30 s at 60 °C for annealing/extension in 40 cycles; and (iii) an imaging step completed by reading in the FAM channel. The entire workflow comprising the three steps takes about 2 h. Data were analyzed using the QIAcuity Software Suite V1.2 and expressed as copies/μL.

cDNA was synthesized from RNA extracts and libraries prepared using the COVIDSeq-Test™ (Illumina, San Diego, USA) with the Artic v4.1 primer sets, following the manufacturer's guidelines and sequenced with 75 bp paired-end reads on an Illumina NextSeq500 system. In each library, two negative and two positive controls were added. Data were processed with the Dragen Lineage pipeline 2.5.6 which provided the Pango lineage, Fasta consensus sequences, and bam files. Viral clades were obtained from consensus sequences with Nextclade (https://clades.nextstrain.org/). Reads were aligned to the reference sequence NC_045512.2. Used parameters were: base calling > 10× and mutation calling in the consensus > 0.5. Coinfection detection was performed manually by the analysis of bam files in the IGV_2.11.1 software and the identification of coinfected variants was performed by the detection of clade-defining mutations (the list on https://covariants.org/variants).

The SARS-CoV-2 genome sequences determined in this study are available at NCBI GenBank under the accession numbers OR335241 to OR335247.

From December 2021 to July 2022, 90% (27/30) of wastewater influent samples tested positive via RT-sdPCR, including 100% of WWTP2, WWTP4, and WWTP5. SARS-CoV-2 was regularly detected in influents from all WWTPs between December 2021 and May 2022. Only three wastewater influent samples tested negative in June 2022 in WWTP4, WWTP5, and WWTP6. SARS-CoV-2 RNA concentrations in positive wastewater samples ranged from 5.3 × 10³ to 9.02 × 10⁶ copies/L (Table 1). The highest SARS-CoV-2 concentration in wastewater samples was detected in WWTP4 at the end of June 2022 and the lowest was detected in WWTP6 at the beginning of June.

SARS-CoV-2 RNA was detected in 42.85% of sewage samples collected at exit points (Supplementary Table S1). Viral loads ranged from 0.054 × 10⁵ to 2.06 × 10⁵ RNA copies/L of wastewater. For the WWTPs where SARS-CoV-2 was detected in treated wastewater samples, the SARS-CoV-2 RNA concentrations were lower than those determined in raw wastewater. Virus RNA dropped to undetectable levels in treated wastewater samples only for WWTP1 and WWTP5 (Supplementary Table S1). Interestingly, all wastewater effluent samples from WWTP1 (optional aerated lagoon) tested negative even when the SARS-CoV-2 viral load was high in wastewater influent samples. In WWTP5 (activated sludge), SARS-CoV-2 RNA was detected in 25% of wastewater effluent samples. In WWTP3 and WWTP4 (both activated sludge), all wastewater effluent samples tested positive when SARS-CoV-2 was detected in raw wastewater.

Selected wastewater samples that tested positive for SARS-CoV-2 were subjected to genome sequence analysis after next-generation sequencing of SARS-CoV-2 amplicons. Amplicon sequencing confirmed the presence of SARS-CoV-2 RNA in all wastewater samples that tested positive with RT-sdPCR. 58% of the sequenced samples reached a genome coverage >50% SARS-CoV-2. Several samples showed low mean amplicon coverage depth and genome coverage, which could be associated with RNA degradation and/or low viral abundance in the wastewater samples.

The present report investigates the presence of SARS-CoV-2 and the dynamics of genomic changes in wastewater sampled from six major urban treatment facilities in Tunisia, reports the correlations with the COVID-19 prevalence data and assesses the effectiveness of wastewater treatment procedures used in the WWTP studied.

From December 2021 to July 2022, SARS-CoV-2 was detected in a majority of the influent samples (n = 27/30) analyzed among six WWTPs. There have been two waves of COVID-19 in Tunisia during the study period. During these waves, the changes in the amount of SARS-CoV-2 RNA in wastewater were consistent with the variations in the number of notified cases of COVID-19. Interestingly, during the second wave, the time between the peak of viral genomes in wastewater and that of COVID-19 cases was 2 weeks while during the first wave, there was no time lag between the two peaks. This difference can be explained by the reconciliation of collection dates of wastewater samples during the second wave. Our data showing a correlation between the concentration of SARS-CoV-2 RNA in wastewater and the number of notified COVID-19 cases are in agreement with previous studies showing a temporal correlation between wastewater SARS-CoV-2 RNA levels and COVID-19 epidemiological features (Trottier et al. 2020; Hasan et al. 2021; Nattino et al. 2022). In addition, our quantitative data indicated that an increase in viral signals could be detected before the increment of clinically diagnosed new COVID-19 cases, as reported earlier (Peccia et al. 2020; Galani et al. 2022). The SARS-CoV-2 RNA concentrations reported in wastewater influent were in the range of 5.3 × 10³ to 9.02 × 10⁶ copies/L. High concentrations of SARS-CoV-2 were detected by the end of June. The range of the detected concentration is higher than that in other studies conducted in Australia, Italy, the United States, Spain, and France where the maximum SARS-CoV-2 RNA concentrations in wastewater influent varied from 1.2 × 10³ to 3.2 × 10⁶ gene copies/L (Ahmed et al. 2020; La Rosa et al. 2020; Nemudryi et al. 2020; Randazzo et al. 2020; Wurtzer et al. 2020; Weidhaas et al. 2021).

Effluents showed a 43% positivity rate, which is high compared with the rates of positive samples of SARS-CoV-2 RNA after secondary treatments in French, Spanish, and Japanese studies (Haramoto et al. 2020; Viveros et al. 2022). However, the ranges of the determined concentrations were comparable. Nevertheless, other studies reported no occurrence of SARS-CoV-2 RNA after secondary treatment (Balboa et al. 2021; Sherchan et al. 2021). These results suggest that the secondary treatment techniques can have a significant impact on virus detection after this treatment. Thus, in our study, all wastewater effluent samples tested negative in WWTP1 using the optional aerated lagoon process even if the SARS-CoV-2 viral load in wastewater influent samples was very high but in the other WWTPs using the activated sludge process, SARS-CoV-2 was detected in treated wastewater samples. Hence, the optional aerated lagoon process adopted at WWTP1 seems to provide better results than the activated sludge process used in the remaining WWTPs. During the study period, two SARS-CoV-2 variants of concern Delta and Omicron (sublineages BA.1, BA.2, and BA.5) responsible for the epidemic outbreaks in the area were identified in wastewater in the study locations.

Delta and Omicron variants have already been detected in wastewater in several countries in the world (Johnson et al. 2022; Reynolds et al. 2022; Silva et al. 2022; Wilhelm et al. 2022) but to the best of our knowledge, this is the first detection of these variants in wastewater in Tunisia.

In December 2021, the SARS-CoV-2 Omicron variant was first reported in Tunisia and rapidly became the predominant circulating variant.

In our study, sublineage BA.1 was detected from December 2021 in wastewater. In February 2022, a mixture of BA.1 and BA.2 was detected in the same sample, suggesting that it was obtained during the transition period from the BA.1 to the BA.2 predominant circulating sublineages as from this date BA.2 displaced BA.1 as the dominant sublineages.

Interestingly, the BA.5 variant was detected in the wastewater sample in February 2022 while this strain was reported later by the Tunisian Department of Health in Tunisian COVID-19 patients. Our results are consistent with previous studies reported that over 50% of the clinical–wastewater sequence pairs show earlier detection in wastewater are from the same date (Joshi et al. 2022; Karthikeyan et al. 2022). Thus, in a recent Indian investigation, the authors identified emerging VOCs in wastewater samples up to 2 months earlier (Lamba et al. 2023).

In conclusion, this study reports the first detection of Delta and Omicron variants in wastewater in Tunisia and identified an increase in viral signal in WWTP influent samples before the increment of clinically diagnosed new COVID-19 cases. Our data also provided evidence that variant BA.5 was detected in samples from the current investigation prior to its inclusion as a variant of concern by the Tunisian National Health authorities, confirming the value of genomic surveillance of wastewater to act as an early warning system.

In addition, our study reports the presence of SARS-CoV-2 RNA in the majority of treated wastewater samples, which can represent a public health risk by waterborne transmission of these viruses mainly a recent study proves the survival of SARS-CoV-2 in secondary effluent (Sherchan et al. 2023).

The authors acknowledge the Directorate of Milieu Hygiene and Environmental Protection at the Tunisian Health Ministry for their collaboration. We thank Dr Dorsaf Ben Malek for the English revision of the manuscript and the African Biotechnology Society for their technical assistance.

Our study is a part of the project: Reinforcement of the National Capacities regarding prevention, coordination, and response to the COVID-19 pandemic in liaison with epidemiological and environmental impacts. This project was funded by the Swiss Agency for Development and Cooperation and executed by the United Nations Development Programme in Tunisia with the collaboration of the Tunisian Ministry of Health and the Ministry of Local Affairs and the Environment. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

I.O., A.H., and M.A. conceived and designed research. I.O., M.B., R.H., and I.N. conducted experiments. I.O., M.A., I.S., M.B., and J.L.B. analyzed data. I.O. wrote the manuscript. M.A., A.H., J.L.B., and M.M. contributed resources, supervision, review, editing, and validation. All authors read and approved the manuscript.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.