Quantification and Trends of Rotavirus and Enterovirus in Untreated Sewage Using Reverse Transcription Droplet Digital PCR

The quantification and trends in concentrations for naturally occurring rotaviruses (RV) and enteroviruses (EV) in untreated sewage in various wastewater systems have not often been compared. There is now greater interest in monitoring the infections in the community including live vaccine efficacy by evaluating untreated sewage. The goals of this study were to 1) survey the concentrations of naturally occurring RV and EV in untreated sewage using a reverse transcription-droplet digital polymerase chain reaction (RT-ddPCR) and 2) investigate the use of a new adsorption elution (bag-mediated filtration system (BMFS) using ViroCap filters) against more traditional polyethylene glycol (PEG) precipitation for virus concentration. Sewage samples were collected from lagoons in Kenya and Michigan (MI), the United States (USA) and from wastewater treatment plants (WWTPs) in the USA. RVs were detected at geometric mean concentrations in various locations, California (CA) 1.31 × 10 genome copies/L (gc/L), Kenya (KE) 2.71 × 10 gc/L and Virginia (VA) 1.48 × 10 gc/L, and EVs geometric means were 3.72 × 10 gc/L (CA), 1.18 × 10 gc/L (Kenya), and 6.18 × 10 gc/L (VA). The mean RV concentrations using BMFS-ViroCap in split samples compared to PEG precipitation methods demonstrated that the levels were only 9% (#s BMFS/PEG) in the Michigan lagoons which was significantly different (p < 0.01). This suggests that RV concentrations in Kenya are around 1.69 × 10 gc/L. Overall, there was no difference in concentrations for the other sampling locations across the methods of virus recovery (i.e., PEG precipitation and HA filters) using one-way ANOVA (F = 1.7, p = 0.2739) or Tukey-Kramer pairwise comparisons (p > 0.05). This study provides useful data on RV and EV concentrations in untreated sewage in Kenya and the USA. It also highlights on the usefulness of the RT-ddPCR for absolute quantification of RV and EV in sewage samples. The BMFS using ViroCap filters while less efficient compared to the more traditional PEG precipitation method was able to recover RVs and EVs in untreated sewage and may be useful in poor resource settings while underestimating viruses by 1 to 1.5 logs.