A one-year study on molecular and epidemiological monitoring and analysis of enteroviruses and waterborne hepatitis throughout various stages of wastewater treatment

Wastewater, a major contributor to waterborne disease outbreaks, is contaminated with various microorganisms, including viruses that cause hepatitis and gastroenteritis. In São José do Rio Preto, São Paulo state, Brazil, samples were collected at three stages - raw sewage (RS), post-anaerobic biological treatment (PABT) and post-chemical treatment (PCT) - over the course of one year (2022-2023) for the detection of Enterovirus (EV), Hepatitis A (HAV), and Hepatitis E (HEV) viruses, with molecular characterization when possible. The 156 collected samples were tested using in-house qPCR for EV and HAV. HEV detection utilized conventional Nested PCR. The findings were correlated with epidemiological data and physicochemical water parameters, considering seasonal and meteorological variations. EV prevalence was 98.1% (RS), 88.5% (PABT), and 42.3% (PCT), while HAV prevalence was 25.0% (RS), 23.1% (PABT), and 13.5% (PCT). HEV was found in two stages, with prevalences of 5.8% (RS) and 3.8% (PABT). EV quantification ranged from 2.20 to 4.45 log 10 GC/mL, and HAV from 2.63 to 4.42 log 10 GC/mL. EV was found in higher concentrations during milder temperatures (autumn and winter) across different wastewater treatment stages ( p ≤ 0.05), while no significance was observed for HAV. EV and HAV showed mean reductions of 1.74 and 2.20 log₁₀, respectively, throughout the treatment process, with the greatest reduction in viral load observed after chemical treatment with chlorine. However, neither virus was completely eliminated, which may pose a potential public health concern. Phylogenetic analysis identified HAV genotype IA and HEV genotype 3, and EV genotype A was detected by Sanger sequencing. The recovery efficiency test using Echovirus 3 showed good recovery of ultracentrifugation method percentages in RS and PABT (68.0% to 48.7%), while PCT samples ranged from 28.1% to 0.0%. No statistically significant differences were found when correlating reported cases of enteric or hepatic disease with the corresponding detected viruses ( p > 0.05). In conclusion, these findings emphasize the need for continuous monitoring of wastewater treatment to effectively assess and mitigate the risk of waterborne diseases.