Abstract
BACKGROUND: Wastewater-based surveillance offers a cost-effective, population-level complement to clinical testing for early detection of infectious disease outbreaks; however, its adoption in low- and middle-income countries remains limited. We conducted a study in Kampala, Uganda, to quantify SARS-CoV-2 RNA in wastewater and evaluate its association with reported clinical cases, thereby strengthening community-level surveillance strategies. METHODS: From March 2023 to May 2024, 244 wastewater samples were collected weekly from four wastewater treatment plants in the Kampala Metropolitan Area, Uganda. SARS-CoV-2 RNA was quantified by RT-qPCR targeting the ORF1ab, N, and E genes using the Novel Coronavirus (2019-nCoV) Real-Time Multiplex RT-PCR kit, with PMMoV as the process control. Concordance of gene detection was assessed using Cohen’s kappa and the proportion of samples in which all targets were detected. SARS-CoV-2 viral concentrations were reported as log₁₀ genomic copies per 100 mL. Facility-level weekly mean ORF1ab concentrations were aggregated into citywide medians and correlated with clinical positivity, with lead–lag analyses to evaluate spatiotemporal associations. RESULTS: Overall SARS-CoV-2 RNA detection was 88.5%, with higher positivity at the Nakivubo wastewater treatment plant inlets (1 and 2) and the Naalya wastewater stabilization pond (both 93.4%) than at the Bugolobi fecal sludge treatment plant (78.7%). All 3 gene targets were detected in 66.4% of samples, with stronger concordance between ORF1ab and E than between ORF1ab and N (κ = 0.68), and facility-specific variability in three-gene detection ranged from 57.4% to 70.5%. Wastewater viral dynamics were characterized by episodic surges rather than sustained peaks, with ORF1ab concentrations ranging from 2.97 to 11.87 log₁₀ GC/100 mL. While same-week wastewater–clinical correlations were weak, lead–lag analysis showed wastewater signals preceded clinical positivity by 2–5 weeks, with the strongest association at a 4-week lead. CONCLUSION: WBS provided early warning of SARS-CoV-2 transmission in Kampala, with clinical positivity preceded by up to 1 month. These findings support the integration of WBS into routine surveillance to enhance outbreak preparedness and response, particularly in resource-limited settings, and to inform public health decision-making. Besides COVID-19, WBS can also track multiple infectious diseases by detecting covert transmission patterns and predicting clinical trends. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12889-026-26267-x.