Comparative Evaluation of SARS-CoV-2 RNA Concentration and Normalization Strategies in Prison Wastewater: Implications for Viral Dynamics in Confined Environments.

BACKGROUND

Wastewater-based epidemiology (WBE) is a valuable population-level surveillance tool for monitoring SARS-CoV-2 circulation. However, evidence on optimal viral concentration approaches in confined institutional settings such as prisons remains limited. This study aimed to compare the performance of Direct Capture (DC) and Electronegative Membrane Filtration (EMF) for SARS-CoV-2 RNA detection in wastewater from a prison facility in Selangor, Malaysia.

METHODS

Composite wastewater samples collected over 18 weeks (April-August 2023; n = 50) were analysed by RT-dPCR targeting the N1 and N2 gene regions, with concentrations normalized to pepper mild mottle virus (PMMoV). DC consistently outperformed EMF across both gene targets. Median concentrations obtained using DC were 11.09 × 103 copies L-1 (N1) and 3.43 × 103 copies L-1 (N2), compared with 0.70 × 103 copies L-1 (N1) and 0.48 × 103 copies L-1 (N2) using EMF. Detection frequencies were higher with DC (N1: 94%, N2: 84%) than with EMF (N1: 88%, N2: 76%). Paired statistical analysis confirmed significant differences between methods (N1: p = 2.3 × 10-7; N2: p = 9.4 × 10-5), and Bland-Altman analysis demonstrated systematic underestimation by EMF (mean bias -1.15 log10 for N1; -0.87 log10 for N2), indicating that the methods are not analytically interchangeable.

CONCLUSIONS

Normalization reduced absolute SARS-CoV-2 RNA concentrations while preserving temporal trends, supporting its use to improve comparability across sampling periods. Overall, these findings demonstrate that DC combined with N1 detection provides a more sensitive and reliable approach for SARS-CoV-2 WBE in confined settings, underscoring the importance of methodological optimization to strengthen early-warning capacity in high-risk environments.