Intraday variability of indicator and pathogenic viruses in 1-h and 24-h composite wastewater samples: implications for wastewater-based epidemiology.

We monitored the density of fecal indicator viruses crAssphage and pepper mild mottle virus (PMMoV) and human pathogen adenovirus (HAdV) in influent from a wastewater treatment plant in Brisbane, Australia in 1-h and 24-h composite samples. Over three days of sampling, the mean concentration of crAssphage gene copies (GC)/mL in 24-h composite samples did not differ significantly (p = 0.72), while for PMMoV GC/mL (p value range: 0.0002-0.0321) and HAdV GC/mL (p value range: 0.72-0.92), significant differences in concentrations, were observed on one day of sampling compared to the other two. For all three viruses the variation observed in 1-h composite samples was greater than the variation observed in 24-h composite samples. For crAssphage, in 54.1% of 1-h composite samples, the concentration was less than that observed in 24-h composite samples; whereas for PMMoV and HAdV the concentration was less in 79.2% and 70.9% of 1-h composites, respectively, compared to the relevant 24-h composite. Similarly, the concentration of crAssphage DNA in 1-h compared to 24-h composite samples did not differ (p = 0.11) while the concentrations of PMMoV (p <0.0001) and HAdV (p <0.0001) in 1-h composites were significantly different from 24-h composites. These results suggest that 24-h composite samples offer increased analytical sensitivity and decreased variability compared to 1-h composite samples when monitoring wastewater, especially for pathogenic viruses with low infection rate within a community. Thus, for wastewater-based epidemiology applications, 24-h composite samples are less likely to produce false negative results and erroneous public health guidance.