Detection of Enteroviruses and SARS-CoV-2 in Tunisian Wastewater.

Monitoring the circulation of enteric viruses in environmental wastewater is a valuable tool for preventing the emergence of waterborne and food-borne diseases in humans. The detection of viruses was performed in five Tunisian wastewater treatment plants, three located in the Grand Tunis City (WWTP 1, WWTP 2, WWTP 3) and two in the Sahel of Tunisia (WWTP 4, WWTP 4), known as very developed and crowded zones, to assess the effectiveness of three biological wastewater treatment procedures namely natural oxidizing lagoons, rotating biodisks procedure, activated sludge procedure, and one tertiary sewage treatment using UV-C254 reactor for this enteric viruses' removal. Thus, 242 sewage samples were collected between June 2019 and May 2020 from different lines of wastewater treatment procedures implemented in the five wastewater treatment plants investigated. SARS-CoV-2 was analyzed using real-time multiplex reverse-transcription polymerase chain reaction (multiplex real-time RT-PCR) and enteroviruses using reverse-transcription polymerase chain reaction (RT-PCR). The enteroviruses detection showed 93% and 73% respective high frequencies only in the two WWTPs of the Grand Tunis (WWTP 1 and WWTP 2). SARS-CoV-2 was detected in 58% of the all wastewater samples collected from the five studied WWTPs with a respective dominance of N gene (47%), S gene (42%), RdRp gene (42%) and at last E gene (20%). These enteroviruses and SARS-CoV-2 detection were revealed in all steps of the wastewater treatment procedures, so poor virological quality is found at the exit of each biological and tertiary step of treatment investigated. For the first time in Tunisia, these results highlighted the enterovirus and SARS-CoV-2 detection with high rates, and the ineffectiveness of the biological and UV-C254 treatment implemented to remove these viruses. The preliminary results of SARS-CoV-2 circulation in Tunisian wastewater confirmed the wide positivity rate underlined by other works worldwide and allowed showing a move towards integrating wastewater as a way for this virus to spread in different areas and environments. So, this last result about SARS-CoV-2 circulation allowed us to caution about the strong probability of diffusion of this hazardous virus through water and sewage; despite its enveloped character and nature, as a labile and sensitive virus in these environments. Thus, establishing a national surveillance strategy is needed to improve the sanitary quality of treated wastewater and prevent public health problems related to these viruses in treated wastewater.

Activated sludge and UV-C254 for Sapovirus, Aichivirus, Astrovirus, and Adenovirus processing.

In this study, the detection rates of four enteric viruses, Human Astrovirus (HAstVs), Aichivirus (AiVs), Human Adenovirus (HAdVs), and Sapovirus (SaVs) are carried out to assess the virological quality of the treated wastewater. A total of 140 samples was collected from wastewater treatment plant WWTP of Tunis-City. Real-time RT-PCR and conventional RT-PCR results showed high frequencies of detection of the four enteric viruses investigated at the entry and exit of the biological activated sludge procedure and a significant reduction in viral titers after tertiary treatment with UV-C254 irradiation. These results revealed the ineffectiveness of the biological activated sludge treatment in removing viruses and the poor quality of the treated wastewater intended for recycling, agricultural reuse, and safe discharge into the natural environment. The UV-C254 irradiation, selected while considering the non-release of known disinfection by-products because of eventual reactions with the large organic and mineral load commonly present in the 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.

The Effectiveness of Activated Sludge Procedure and UV-C254 in Norovirus Inactivation in a Tunisian Industrial Wastewater Treatment Plant.

The molecular detection of Norovirus GI and Norovirus GII in the Tunisian industrial wastewater treatment plant of Charguia I was conducted to test the effectiveness of secondary biological treatment using the activated sludge procedure and the UV-C254 tertiary treatment radiation using a UV disinfection prototype to upgrade the quality of the purified wastewater. A total of 140 sewage samples were collected from the two lines of sewage treatment procedures. Norovirus GI and Norovirus GII have been found and quantified using Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR) in 66.4 and 86.4% of the collected wastewater samples. The Norovirus GI and GII mean concentration values got in the treated effluents showed a significant decrease of Norovirus viral content rates detected from upstream to downstream of activated sludge procedures and at the output of UV-C254 treatment. These findings characterise and denote for the first time the effectiveness of biological and UV-C254 treatment for Norovirus GI and Norovirus GII removal in Tunis City, northern Tunisia. Also, these data underlined the fact that purified sewage makes up a route of gastroenteritis virus transmission and a cause of viral gastroenteritis associated with water-borne and food-borne outbreaks.

The performance of biological and tertiary wastewater treatment procedures for rotaviruses A removal.

Enteric viruses, generally found in sewage, are recognized as the main cause of waterborne and foodborne public health outbreaks. Among leading enteric viruses, the Rotavirus A (RVA) detection in wastewater appeared to be a novel approach to monitor the emergence of these viruses in some countries where the viral gastroenteritis surveillance is almost absent such as in Tunisia. The RVA detection and quantification in an industrial sewage purification plant of Charguia I (Tunis, Tunisia) were achieved to evaluate the performance of activated sludge procedures coupled to a macrofiltration monolamp ultraviolet irradiation type C (UV-C254) disinfection reactor. This UV-C254 system was preceded by a fiberglass cartridge filter system with an average porosity of 45 µm to clarify the water and thus increase its UV transmittance. A total of 140 composite sewage samples was collected from this line of treatment and analyzed for RVA detection. The detection and the viral load quantification of RVA were performed using real-time reverse transcription polymerase chain reaction (RT-PCR). The virological results showed in general that RVA were detected at high frequency of 98% (137/140). In fact, the RVA detection rates at the exit of the two studied wastewater treatment were about 100% at the exit of the activated sludge procedure. It means that all wastewater sampled at this last step of treatment was positive for RVA detection. On the other hand, 92.5% of the wastewater samples taken at the exit of the monolamp UV-C254 reactor were positive for the RVA. However, the RVA quantification results expressed as viral load showed a significant reduction in the means of RVA viral loads at the exit of the biological activated sludge procedure and the tertiary UV-C254 treatment, showing in general an improved treated wastewater virological quality. Therefore, the RVA load removal rates recorded at the two successive stages of treatment, the activated sludge and the UV-C254 treatment, were around 85% and 73%, respectively, as compared to the one with 100% registered for the raw wastewater. In addition, good physical-chemical and bacteriological qualities of the treated sewage were found at the exit of the two considered wastewater treatment procedures. The present investigation represents the first Tunisian environmental report showing the good effectiveness and performance of the biological and the tertiary treatments for RVA removal. Therefore, an improved and an optimized tertiary disinfection treatment was needed since it could be a good means for getting better viral water quality and for minimizing the transmission and dissemination of human infectious viral diseases.