Monitoring Human Viral Pathogens Reveals Potential Hazard for Treated Wastewater Discharge or Reuse

Abstract

Wastewater discharge to the environment or its reuse after sanitization poses a concern for public health given the risk of transmission of human viral diseases. However, estimating the viral infectivity along the wastewater cycle presents technical challenges and still remains underexplored. Recently, human-associated crAssphage has been investigated to serve as viral pathogen indicator to monitor fecal impacted water bodies, even though its assessment as biomarker for infectious enteric viruses has not been explored yet. To this end, the occurrence of potentially infectious norovirus genogroup I (GI), norovirus GII, hepatitis A virus (HAV), rotavirus A (RV), and human astrovirus (HAstV) along with crAssphage was investigated in influent and effluent water sampled in four wastewater treatment plants (WWTPs) over 1 year by a PMAxx-based capsid integrity RT-qPCR assay. Moreover, influent and effluent samples of a selected WWTP were additionally assayed by an in situ capture RT-qPCR assay (ISC-RT-qPCR) as estimate for viral infectivity in alternative to PMAxx-RT-qPCR. Overall, our results showed lower viral occurrence and concentration assessed by ISC-RT-qPCR than PMAxx-RT-qPCR. Occurrence of potentially infectious enteric virus was estimated by PMAxx-RT-qPCR as 88–94% in influent and 46–67% in effluent wastewaters with mean titers ranging from 4.77 to 5.89, and from 3.86 to 4.97 log10 GC/L, with the exception of HAV that was sporadically detected. All samples tested positive for crAssphage at concentration ranging from 7.41 to 9.99 log10 GC/L in influent and from 4.56 to 6.96 log10 GC/L in effluent wastewater, showing higher mean concentration than targeted enteric viruses. Data obtained by PMAxx-RT-qPCR showed that crAssphage strongly correlated with norovirus GII (ρ = 0.67, p < 0.05) and weakly with HAstV and RV (ρ = 0.25–0.30, p < 0.05) in influent samples. In effluent wastewater, weak (ρ = 0.27–0.38, p < 0.05) to moderate (ρ = 0.47–0.48, p < 0.05) correlations between crAssphage and targeted viruses were observed. Overall, these results corroborate crAssphage as an indicator for fecal contamination in wastewater but a poor marker for either viral occurrence and viral integrity/infectivity. Despite the viral load reductions detected in effluent compared to influent wastewaters, the estimates of viral infectivity based on viability molecular methods might pose a concern for (re)-using of treated water.

Wastewater-based surveillance for tracing the circulation of Dengue and Chikungunya viruses

“…Our study's crAssphage concentration results align with findings from previously published studies. 22,23 The RNA from all viruses under scrutiny was consistently detected in raw urban wastewater samples (see Figure 1). DENV was consistently present throughout the sampling period, while CHIKV detection was more prevalent in 2023, particularly during March and April.…”

“…Our study’s crAssphage concentration results align with findings from previously published studies. 22,23…”

Predicting the dispersal of SARS-CoV-2 RNA from the wastewater treatment plant to the coast

“…Therefore, the behavioural dynamics and transport modelled in the two rivers in this study could be transferable to the dynamics of other enveloped viruses. Non-enveloped RNA viruses, such as norovirus and rotavirus, which are two of the most common causes of viral gastroenteritis, are also commonly discharged from WWTPs into receiving waters ( Cuevas-Ferrando et al, 2022 ). Such enteric viruses can remain stable in the environment for extended periods and pose public health risk to those exposed to contaminated water ( Dean and Mitchell, 2022 ) – in which case model insights from the T 90 = 35 days simulations are particularly pertinent.…”

“…The dashed red line shows the mean daily surface elevation and the solid red bounding lines plot the daily max and minimum surface elevations. commonly discharged from WWTPs into receiving waters (Cuevas-Ferrando et al, 2022). Such enteric viruses can remain stable in the environment for extended periods and pose public health risk to those exposed to contaminated water (Dean and Mitchell, 2022) in which case model insights from the T 90 ¼ 35 days simulations are particularly pertinent.…”

Genome Analysis of Epsilon CrAss-like Phages

“…This fact can be partly explained by the association of Bacteroides with the diet of urban populations [ 14 ]. Due to the high prevalence of crAssphages in the human intestine, it is currently proposed to monitor their levels in treated wastewater to detect contamination with human feces [ 15 , 16 , 17 ].…”