Wastewater surveillance is useful for monitoring the prevalence of hepatitis A virus (HAV). We developed and optimized HAV detection and quantification methods for wastewater samples, and applied them to samples collected through a national wastewater surveillance program. Previously identified 5-untranslated region-targeting primers and probes were used to develop the assay. Serial dilutions of HAV-positive clinical samples were used to validate and determine limits of quantification (LOQ). Retrospective testing of weekly wastewater samples collected through the SARS-CoV-2 wastewater surveillance program at 26 sites in Gauteng (August 2021 to March 2024) were undertaken using ultrafiltration-based concentration, and nucleic acids were extracted using the KingFisher Flex purification system with a wastewater isolation kit. A digital PCR assay was used for HAV detection and quantification (as genome copies/L). Clinical data from the Surveillance Database Warehouse of the National Health Laboratory Service were compared with wastewater data, epidemiological week-wise and district-wise, to determine correlations between the datasets. Based on the validation results, one partition on the digital PCR (dPCR) platform was equivalent to an LOQ of 0.4 genome copies/L. In total, 2013 wastewater samples were tested, of which 349 were positive for HAV (17.3%), wherein the majority (304, 87.1%) had the lowest HAV concentration (2.0-2.7 gc/L, 1-5 partitions), followed by 20 samples (5.7%) with concentrations of 2.8-3.0 gc/L (6-10 partitions). HAV was detected in 17.1% (241/1170) of the Gauteng samples, and a 26.1% correlation between anti-HAV IgM in clinical samples and HAV in wastewater samples was detected. We successfully developed and optimized a dPCR method to detect and quantify HAV in wastewater samples, and determined its LOQ. Further analysis is required to compare the wastewater data with clinical surveillance data to facilitate appropriate interpretation of the results.

The authors have declared no competing interest.

The funder: Bill and Melinda Gates Foundation (https://www.gatesfoundation.org/), grant (INV-049271), and (INV-050051); MY and KM were the PIs. The funders had no role in the data collection and analysis, decision to publish, or preparation of the manuscript

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The NICD conducts all routine clinical surveillance, including NMC surveillance, in a protocol reviewed and approved by the University of the Witwatersrand Human Research Ethics Committee (HREC) M210752 and under the legal authority of the National Health Act (no. 61 of 2003). Wastewater sampling and testing were performed using an HREC system (M230828). Informed consent was not required as we only used anonymized data

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