Decay dynamics of hepatitis C virus RNA in wastewater under variable environmental conditions

Hepatitis C virus (HCV) is a bloodborne, enveloped RNA virus that chronically infects over 50 million persons and causes significant mortality due to hepatocellular carcinoma and cirrhosis. Although wastewater-based epidemiology (WBE) has been widely used for enteric viruses, such as hepatitis A and hepatitis E, HCV surveillance via WBE remains underexplored, particularly for high-burden urban settings like Baltimore, Maryland. This study investigates the decay kinetics and environmental persistence of HCV RNA in three wastewater matrices (raw, autoclaved, and filtered) at two temperatures (25 °C and 37 °C) to assess the feasibility of HCV surveillance through WBE. Laboratory microcosms were prepared with high (10⁹ IU) and low (10³ IU) concentrations of HCV RNA spiked into wastewater matrices. Samples were incubated at target temperatures, and viral RNA degradation was monitored over 5 days using RT-qPCR. First-order decay constants (k) and time to 90% reduction (T₉₀) were calculated. Raw wastewater exhibited the highest RNA decay rates (k = 0.1069-day¹ at 25 °C), with the shortest half-life, attributed to enzymatic and microbial degradation. Autoclaved and filtered matrices showed significantly slower decay, particularly at lower temperatures. Temperature influenced RNA degradation, but this effect was secondary to the matrix composition. RT-qPCR inhibition was minimal in treated samples. HCV RNA stability was strongly influenced by temperature and wastewater treatment, with the most rapid degradation observed in untreated wastewater. The observed differences between raw and autoclaved/filtered matrices suggest that microbial and enzymatic activity likely contribute to RNA instability. These findings emphasize the need for high-frequency sampling and decay-adjusted modeling for accurate HCV surveillance in WBE. This study supports the integration of HCV testing into wastewater-based monitoring, particularly in communities with high HCV prevalence.