Comparative Evaluation of SARS-CoV-2 RNA Concentration and Normalization Strategies in Prison Wastewater:...

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.

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.

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.