Wastewater-based Surveillance for Hepatitis A and C in an Inflow- and Infiltration-Impacted Rural System

Abstract

Wastewater-based surveillance (WBS) refers to the systematic sampling and testing of wastewater to analyze different targets, such as pathogens, for the benefit of public health. The use of WBS can be complementary to clinical data to analyze and track pathogens within a community, and it also has the potential to supplement clinical data in communities where members have difficulty accessing regular health care. Most WBS studies are conducted in urban settings. Rural-urban health disparities are prevalent nationwide and are particularly acute in the Appalachian Region. There are several risk factors in this region for hepatitis infection. Hepatitis is an inflammation of the liver which can develop into chronic hepatitis and is caused by one or more of the five primary strains (A-E). Hepatitis A (HAV) is typically spread via the fecal oral route and consumption of contaminated food and/or water. Hepatitis C (HCV) is primarily a bloodborne virus, often transmitted through intravenous drug use. In many under-funded and smaller rural sewer systems, dilution and other effects from inflow and infiltration (IandI) may have substantial impacts on pathogen detection. The objective of this study was to assess the potential for WBS in small, rural systems influenced by IandI to detect and track HAV and HCV as representative viruses that cause relatively rare infections. In a small, rural community (<3,000 people) in southwest Virginia, wastewater composite and grab samples (53 total) were collected at monthly intervals over a 12-month period (March 2024 – February 2025) at the wastewater treatment plant (WWTP) influent and five up-sewershed sites. In-situ testing of physicochemical parameters (pH, conductivity, dissolved oxygen, temperature) was done immediately after sample collection. Daily precipitation, daily temperature, and daily WWTP influent flow were obtained from the WWTP staff. Chemical oxygen demand (COD) was measured within 24 hours of sample collection. Droplet digital PCR (ddPCR) was performed in triplicates to quantify viral targets HAV, HCV, SARS-CoV-2, and the human fecal indicator crAssphage. The likelihood of missed detection at the WWTP influent for HAV and HCV compared to the sub-sewershed node level was evaluated and we assessed whether other wastewater parameters may be used to inform the likelihood of HAV and HCV detection at up-sewershed nodes or at the WWTP influent. COD data collected over the study period aligned with COD trends and up-sewer sampling site IandI categorizations (limited, precipitation-driven, permanent) established in previous work (2022-2023) at the same study site. Two different detection criteria were used to analyze positive detections of HAV and HCV. We found that with both conservative and less conservative detection criteria HAV and HCV were detected at sub-sewershed nodes, but not always at the WWTP influent. Using conservative detection criterion, there were 3 sampling campaigns (3/8) with positive HAV detections at up-sewershed nodes but not at the WWTP, and no detection for HCV; using a less conservative detection criterion, HCV was detected at the influent but not up-sewer and vice versa on different sampling events. We also observed spatial patterns that aligned with hepatitis etiology at different up-sewer sites. As far as we are aware, this is the first WBS study to measure HCV in a rural setting, and the first to assess IandI impacts on HAV or HCV in any setting. Our findings suggest in smaller sewer systems with IandI impacts, wastewater sampling only at the WWTP may not provide a reliable metric for pathogen circulation trends for less commonly circulating viruses. More research is needed to understand the extent to which false negatives at WWTP influent may or may not be an issue for systems with less IandI and for larger and urban systems.