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

Wastewater-based genomic surveillance can improve community prevalence estimates and identify emerging variants of pathogens. Wastewater influents and treated effluents from six wastewater treatment plants (WWTPs) in Tunisia were analyzed between December 2021 and July 2022. Wastewater samples were analyzed with reverse transcription solid digital PCR (RT-sdPCR) and whole-genome sequencing to determine the amount of SARS-CoV-2 RNA and assign SARS-CoV-2 lineages. The virus variants detected in wastewater samples were compared with COVID-19 prevalence data. The quantitative results in wastewater influents revealed that viral RNA concentrations at the treatment plants corroborate with locally reported clinical cases and show an increase before the increment of clinically diagnosed new COVID-19 cases between April and July 2022. Delta and Omicron variants were identified in the Tunisian wastewater. Interestingly, the presence of variant BA.5 was detected in samples prior to its inclusion as a variant of concern (VOC) by the Tunisian National Health Authorities. SARS-CoV-2 was detected in wastewater effluents, indicating that the wastewater treatment techniques used in the majority of Tunisian WWTPs are inefficient in removing the virus traces. This study reports the first identification of SARS-CoV-2 VOCs in Tunisian wastewater samples.

Microbiological and physicochemical quality enhancement of treated wastewater using raw and chemically modified clays from Sidi Bouzid region, Tunisia.

Environmental discharge of wastewater represents a source of chemical and biological pollutants. This study firstly evaluates the microbiological and physicochemical quality of treated wastewaters collected from two wastewater treatment plants (WWTPs) located in two different Tunisian cities namely Sidi Bouzid (SB) and Gafsa (G). Then, the capacity of three raw and acid/base-activated local clays to enhance the quality of wastewaters was assessed. The results indicate that the quantities of enteric bacteria (oscillating from 1.381 × 103 to 1.4 × 108 CFU/100 mL), fungi (between 1.331 × 103 and 1.781 × 104 CFU/100 mL), as well as SARS-CoV-2 (between 4.25 × 103 and 5.05 × 105 CFU/100 mL) and Hepatitis A virus RNA (form 4.25 × 103 to 7.4 × 104 CFU/100 mL) detected in effluent wastewaters were not in compliance with the Tunisian standards for both studied WWTPs. Likewise for other indicators such as electrical conductivity (ranging 4.9-5.4 mS/cm), suspended matter (145-160 g l-1), chemical oxygen demand (123-160 mg l-1), biological oxygen demand 5 (172-195 mg l-1), chloride, Total Kjeldahl nitrogen (TKN) and phosphorus contents (710, 58-66 and 9.47-10.83 mg l-1 respectively), the registered values do not agree with the set standards established for wastewater treatment. On the other hand, the pH values fitted (oscillating from 6.86 (at G) to 7.24 (at SB) with the Tunisian standards for both WWTPs. After treatment, wastewaters showed better values for the microbiological parameters, especially for the clays designed as AM and HJ1, which eliminated 100% of viruses. In addition, when acid-activated AM clays were applied, a marked improvement in the quality of physicochemical parameters was obtained, especially for suspended matter (2 and 4 g l-1 for SB and G, respectively), TKN (5.2 (SB) and 6.40 (G) mg/l), phosphorus (1.01 (SB) and 0.81 (G) mg/l). Our results open perspectives for the possibility of efficiently using these specific clays in the enhancement of the quality of treated wastewaters.

Evaluation of three viral concentration methods for detection and quantification of SARS-CoV-2 in wastewater.

Wastewater-based epidemiology (WBE) could be useful as an early warning system for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic spread. Viruses are highly diluted in wastewater. Therefore, a virus concentration step is needed for SARS-CoV-2 wastewater detection. We tested the efficiency of three wastewater viral concentration methods: ultrafiltration (UF), electronegative membrane filtration and aluminum hydroxide adsorption-elution. We spiked wastewater with inactivated SARS-CoV-2 and we collected 20 other wastewater samples from five sites in Tunisia. Samples were concentrated by the three methods and SARS-CoV-2 was quantified by reverse transcription digital PCR (RT-dPCR). The most efficient method was UF with a mean SARS-CoV-2 recovery of 54.03 ± 8.25. Moreover, this method provided significantly greater mean concentration and virus detection ability (95%) than the two other methods. The second-most efficient method used electronegative membrane filtration with a mean SARS-CoV-2 recovery of 25.59 ± 5.04% and the least efficient method was aluminum hydroxide adsorption-elution. This study suggests that the UF method provides rapid and straightforward recovery of SARS-CoV-2 in wastewater.

Inactivation of Hepatovirus A in wastewater by 254 nm ultraviolet-C irradiation.

Hepatovirus A is known as a waterborne and foodborne virus that can be transmitted from one person to another through contaminated water and raw food. Therefore, it is necessary to survey the circulation of this type of enteric virus in the wastewater to prevent prospective outbreaks. Wastewater samples collected from WWTP El Menzeh I and Charguia I have been the subject for physicochemical, bacteriological (MPN) and virological analyses. Hepatovirus A (HAV) detection was carried out using the standard reverse transcription-polymerase chain reaction (RT-PCR). Hepatovirus A was detected respectively in 62% (63/102) and 66% (92/140) of the collected wastewater samples at El Menzeh I and Charguia I WWTPs. The treated effluent by natural oxidizing lagoon procedure was characterized by a poor physical-chemical and virological qualities but with excellent bacteriological quality. Consequently, this effluent is not suitable to be recycled and reused in agriculture or even dismissed in the environment. The treated sewage by activated sludge and rotating biodisk procedures turned out to be of a very good physical-chemical quality but with a poor bacteriological and virological quality. After tertiary UV-C254 nm irradiation, the faecal indicator bacteria concentration was mostly reduced and removed. These findings confirmed the need for improvement and upgrade of the treatment processes used in these two studied sewage purification plants and the necessity of implementation and establishment of a proper national virological standard to control the circulation rates of enteric viruses in Tunisian municipal wastewater.