Statewide multi-year wastewater sequencing reveals dual origins of HIV-1 signal.

Human immunodeficiency virus 1 (HIV-1) is a retrovirus which has infected 90 million people and resulted in over 40 million deaths. Despite advances in diagnostics, treatment, and prophylaxis, HIV-1 continues to spread due to undiagnosed and untreated infections. Traditional monitoring methods are ineffective when access to testing is limited or people do not seek care, particularly given the long period between infection and symptom onset, allowing undetected transmission to continue. Here, we use a hybrid-capture sequencing approach to track HIV-1 signal in municipal wastewater in 15 different cities over nearly 3 years. We obtain near-complete genomic coverage of HIV-1, enabling detailed genomic analysis. Surprisingly, there are a substantial number of research-associated retroviral vector sequences recovered. Using computational competitive mapping, we identify specific genomic regions that differentiate authentic HIV-1 from vector-derived inputs. In an exploratory analysis of sites with available clinical data, wastewater-derived circulating HIV-1 reads show a positive correlation with community-level HIV diagnosed prevalence that was robust to exclusion of individual high-prevalence sites. This study identifies lentiviral vector contamination as a confounding factor in wastewater HIV-1 detection, recovers authentic circulating HIV-1 signal through an original classification framework, and provides initial evidence that the resulting signal tracks community HIV burden.