First detection of hepatitis E virus in wastewater in Costa Rica

Hepatitis E virus (HEV) is an important cause of acute viral hepatitis worldwide, with both human-restricted (genotypes 1 and 2) and zoonotic lineages (genotypes 3 and 4). However, information regarding HEV circulation in Central America remains limited. In this study, wastewater-based epidemiological surveillance was conducted in San José, Costa Rica, during 2025 to investigate the presence of HEV in the main wastewater treatment plant serving approximately one million inhabitants. A total of 54 influent and effluent samples were analyzed. HEV RNA was not detected by quantitative RT-PCR; however, RT-nested PCR targeting the ORF1 region identified three positive influent samples. Sequencing yielded two high-quality partial ORF1 sequences suitable for phylogenetic analysis. Both Costa Rican sequences clustered together and showed a close relationship to genotype 3 sequences. To our knowledge, this represents the first report of HEV detected in wastewater in Costa Rica and the first scientific report of environmental detection in Central America. These findings highlight the value of wastewater-based surveillance for identifying circulating viral pathogens in regions where routine clinical testing is limited.