Optimizing RT-qPCR Detection of Enveloped and Non-enveloped Viruses in Wastewater: A Comparative Study in Grenada

Abstract Introduction/Objective Wastewater-based epidemiology (WBE) is an effective methodology to monitor the community spread of pathogens and has successfully been used to identify potential outbreaks. Most standardized methodologies used in WBE for the detection of viruses have been validated for non-enveloped viruses; therefore, there is a need for the development of a standardized protocol for the detection of both enveloped (such as SARS- CoV-2) and non-enveloped viruses (such as Hepatitis A). Wastewater seeded with Pseudomonas syringae bacteriophage (Φ6), an enveloped virus surrogate, and coliphage MS2, a non-enveloped virus surrogate, were used to evaluate the efficiency of four different aluminium flocculation methods. Untreated wastewater spiked with known amounts of surrogates was used as a control for viral recovery. Methods/Case Report Four different viral concentration methods were used. Method 1: AlCl3 flocculation at pH 3.5 followed by centrifugation, resuspension with 10% beef extract and filtered with a 0.22 μm filter. Method 2: AlCl3 flocculation at pH 6.0 followed by room temperature agitation, centrifugation, and resuspension with 3% beef extract. Method 3: Mixed method protocol-AlCl3 flocculation at pH 3.5 followed by room temperature agitation, centrifugation, resuspension with 3% beef extract. Method 4: Mixed method protocol-AlCl3 flocculation at pH 6.0 followed by centrifugation, resuspension with 10% beef extract filtered with a 0.22 μm filter. Viral RNA was extracted after concentration, and reverse-transcription quantitative polymerase chain reaction (RT- qPCR) was performed. Results (if a Case Study enter NA) The average Ct reduction of untreated wastewater compared to spiked was calculated for each method. The highest recovery of both Φ6 and MS2 was obtained using Method 3 (mixed protocol) with a significant difference at 95% confidence in the recovery of Φ6 (p= 0.0029) and MS2 (p= 0.0225). Conclusion Previous results from our lab demonstrate the importance of pH during the flocculation step of viral concentration using different methods of concentration. Here, the results suggest that the increased contact time between virus and the flocs is also critical. This can facilitate the implementation of WBE in Grenada to monitor and track potential viral outbreaks of both enveloped and non-enveloped viruses such as SARS-CoV-2 and Hepatitis A.