Publication date: May 30, 2025

Wastewater surveillance is an economical approach to track community level pathogen prevalence and outbreak monitoring. Monitoring pathogens of public health concern in military installation wastewater can help mitigation efforts to protect service members. We used laboratory-prepared samples seeded with heat-inactivated SARS-CoV-2 and optimized two methods for wastewater SARS-CoV-2 surveillance. The first one is based on polyethylene glycol/sodium chloride precipitation to which fetal bovine serum (FBS) was added during precipitation (FBS-PEG/NaCl) followed by nucleic acid extraction and SARS-CoV-2 N1 and N2 specific RT-qPCR. This FBS-PEG/NaCl method (method-1) produced a visible pellet and improved recoveries (∼ 84. 13 +/- 9. 12 % for N1 and 71. 59 +/- 9. 77 % for N2, with 300 μL of added FBS) relative to standard PEG/NaCl method (no FBS; ∼7. 98 +/- 8. 24 % for N1 and ∼ 7. 42 +/- 5. 21 % for N2). The second one is a magnetic bead-based improved direct extraction method (method-2) wherein we showed that addition of carrier RNA (polyadenylic acid or poly-A) during extraction improved viral RNA recovery relative to direct extraction without poly-A. We evaluated the analytical sensitivity (process limit of detection; PLOD) and process efficiency of method-1 and method-2. The PLOD was 2,500 genome copies (GC)/L for method-1 and 12,500 GC/L for method-2. Controlled samples with serial dilutions of virus showed that method-1 is as effective in recovering the virus as method-2 at all concentrations of the virus tested. SARS-CoV-2 temporal trends were analyzed in 52 wastewater samples by method-2 of which the first 22 were also analyzed by method-1. SARS-CoV-2 trends correlate well in both approaches while the magnitude (copies/L) was much higher with method-2 (N1: 0. 25-2. 3 log; N2: 0. 058-1. 89 log) than with method-1. Possible reason for lower recoveries by method-1 with wastewater samples and the advantages of using controlled samples for method optimization are discussed. Better recoveries and higher throughput suggest improved direct extraction (method-2) is advantageous for wastewater SARS-CoV-2 surveillance.

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Original Article