Influence of storage conditions and sample processing on viral particle recovery from untreated municipal wastewater
Authors:   , Subroyen Sueyanka 
Published: September 2024

The emergence and re-emergence of several epidemic and pandemic related pathogens has highlighted the need for the development and implementation of rapid and sensitive disease surveillance tools. Wastewater-based epidemiology (WBE) is one such environmental surveillance tool that has been adapted to provide a near real-time reflection of a population’s health dynamics through detecting and quantifying biomarkers from untreated wastewater. This information can be used to monitor disease progression, detect infection hotspots as well as emerging variants. The success of WBE, however, is determined by the accuracy of pathogen detection and quantification methods. Various factors, such as transportation, storage conditions, and wastewater characteristics can affect the quantification, leading to unreliable results. This study aimed at investigating the effects of storage temperatures on SARS-CoV-2 and influenza A degradation in raw wastewater samples and extracted nucleic acids. Additionally, different pre-treatment strategies for improving viral recovery from wastewater solids were also examined as part of the optimization process. Municipal wastewater influent was collected and stored at 25°C, 4°C, -20°C, and -80°C for 84 days. A control sample (spiked with attenuated viruses) was also stored under the same conditions. The viral RNA was extracted and quantified from both the samples (stored wastewater and control) every two weeks. Additionally, the physicochemical characteristics of the wastewater at different temperatures were evaluated for their possible impact on degradation. Supernatant samples and solid particles were used separately for RNA extraction and quantification to assess viral attachment to solid particles. Degradation of influenza A was observed in all samples with the highest at ±25°C in the supernatant (88.89%) and pellet (83.47%) of the wastewater influent, ±25°C in the spiked supernatant (90.69%) and 4°C in the spiked pellet (92.64%). The highest degradation of SARS CoV-2 was observed at ±25°C in the supernatant (94.1%) and pellet (92.66%) of the wastewater influent, 4°C in the spiked supernatant (95.57%) and -20°C in the spiked pellet (50.79%). The lowest degradation of SARS-CoV-2 in the spiked pellet was observed at ±25°C (44.82%) and 4°C (31.53%). This may be indicative of viral adhesion to wastewater solids. The assessment of the physicochemical characteristics indicated of changes in stored wastewater samples. Salinity, DO, pH, COD, TS, and TFS were correlated with viral degradation. pH was also found to be correlated with viral particles attaching to wastewater solids. The effect of storage temperature on stored RNA was also studied using the extracted RNA for a period of 168-days. The degradation between -20°C and -80°C was significantly different for RNA storage. The lowest degradation of SARS-CoV-2 occurred at -80°C (32.51% - wastewater influent; 33.42% - viral controls). In the case of influenza A, the lowest degradation occurred at -80°C for wastewater influent (43.28%) and -20°C for viral controls (36.02%). Statistical analysis conducted comparing storage of wastewater influent to extracted RNA indicated that degradation was higher in the wastewater samples. Additionally, to enhance viral recovery from wastewater solids, sodium pyrophosphate and ultrasonication were explored as pre-treatments strategies. SARS-CoV-2 concentrations in the supernatant were significantly increased between 3.30 - 35.65% by ultrasonication at frequencies ranging from 4 to 16 kHz, indicating that significant amounts of RNA may be attached to solid particles based on the contact time. These results highlight the importance of additional pre-treatment methods for maximizing RNA recovery from wastewater samples. The findings of this research may contribute significantly to the improvement of WBE detection methods for disease surveillance.