Prevalence of enteric viruses in wastewater in Egypt after the COVID-19 pandemic

Prevalence of enteric viruses is affected by hygienic status, therefore, their infection rates increase in countries with low hygienic levels (Payment 2001). According to the WHO, enteric viruses such as HAV, HEV, rotavirus, adenovirus, norovirus, and others are classified as pathogens having moderate to high harmful effects on human health (WHO 2011). Reports detected those viruses in Egypt (Shaheen et al. 2018; Elmahdy et al. 2020).

Usage of wastewater in crops irrigation makes them a source of enteric viral infection (Gerba & Choi 2006) and this was shown in different reports in Egypt with the presence of human adenovirus and rotavirus in green onion and lettuce (Shaheen et al. 2019), presence of HAV and human norovirus in watercress, strawberry, and coriander (Elmahdy et al. 2022).

Following hygienic habits such as carefully washing fruits and vegetables before eating, washing hands with soap and water, cleaning surfaces from dust, and using water filters in houses can help protect humans from enteric virus infections.

After the outbreak of the COVID-19 pandemic, many people tended to follow announced hygienic precautions to protect themselves from SARS-CoV-2 infection including not only the above-mentioned precautions but in addition many people soak fruits and vegetables in water containing vinegar before eating, give up buying street food and boil drinking water, which can all protect from enteric viruses transmission.

Thus, here we aimed to study if safety precautions against SARS-CoV-2 indirectly affected the prevalence of enteric viruses among Egyptians.

One litre of wastewater was collected weekly through the period between August 2021 and March 2022 (21 samples in total) from the Zenin wastewater treatment plant (WWTP) which is one of the largest WWTPs in the Giza government, Egypt. It receives 275,000–550,000 m³/day with an average of 385,000 m³/day of wastewater flow (Latif 2022).

50 mL of 1 M aluminium chloride was added to each wastewater sample to give a final concentration of 0.05 M followed by adjusting pH to 3.5 with 1 N HCl. Samples were filtered through 0.45 μm nitrocellulose membrane then 200 mL 0.5 mM H₂SO₄, pH 3.0 passed after to ensure viral particle retention and eliminate all bio-solids. The membrane was removed from the holder and soaked in a Petri dish containing 11 mL of 1 mM NaOH; pH 10.5 for 10 min. Later, the upper surface of the membrane was scraped to elute the viruses. Volumes of 50 μL 50 mM H₂SO₄ and 50 μL 100X 1 nM Tris-EDTA, pH 8.0 were added to neutralize the elute and samples were stored at −20 °C until use (Katayama et al. 2002).

A volume of 140 μL of concentrated sample was subjected to viral RNA/DNA extraction (Qiagen, Germany) followed by reverse transcription (SuperScript III Reverse transcriptase, Invitrogen, USA) for RNA viruses. Quantitative RT-PCR was performed using SYBER green master mix (Luna, New England) and in-house designed primers (Table 1). Reaction mixture was subjected to the following temperature conditions, initial denaturation: 95 °C for 60 s, denaturation at 95 °C for 15 s, annealing: 55 °C for adenovirus and 57 °C for both HAV and rotavirus for 10 s, extension at 60 °C for 15 s for 40 cycles (Buston et al. 2009).

Since the beginning of the COVID-19 pandemic, many people cared more about the hygienic status of their children, family, and houses and stopped many habits such as eating food from shops or street food, eating fruits and vegetables washed only with water and carefully washed their hands with soap and water and used disinfectants to sterilize many products before taking them inside their houses to protect themselves from SARS-CoV-2 infection. As the prevalence of enteric viruses in a given population depends greatly on hygienic status, we believe that protective precautions against SARS-CoV-2 must have an indirect effect on decreasing their infection rate.

To prove that, 21 samples were collected weekly through the period from August 2021 to March 2022 covering periods of increasing infection rates among Egyptians during the summer months and school season.

Samples were collected from Zenin WWTP, which is one of the largest WWTP in Egypt and receives a huge amount of wastewater per day, and thus detection rate of viruses in it can represent a wide sector of other WWTPs.

Safety precautions against SARS-CoV-2 lead indirectly to a remarkable reduction in the prevalence rates of adenovirus, HAV, and rotavirus prevalence in Egypt.

This work was funded by the National Research Center (Grant number: E-120806).

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

The authors declare there is no conflict.