Human norovirus (HuNoV) GII RNA in wastewater solids at 145 United States wastewater treatment plants: comparison to positivity rates of clinical specimens and modeled estimates of HuNoV GII shedders

Abstract

Background: Human norovirus (HuNoV) is a leading cause of disease globally, yet actual incidence is unknown. HuNoV infections are not reportable in the United States, and surveillance is limited to tracking severe illnesses or outbreaks. Wastewater monitoring for HuNoV has been done previously and results indicate it is present in wastewater influent and concentrations are associated with HuNoV infections in the communities contributing to wastewater. However, work has mostly been limited to monthly samples of liquid wastewater at one or a few wastewater treatment plants (WWTPs).

Objective: The objectives of this study are to investigate whether HuNoV GII preferentially adsorbs to wastewater solids, investigate concentrations of HuNoV GII in wastewater solids in wastewater treatment plants across the county, and explore how those relate to clinical measures of disease occurrence. In addition, we aim to develop and apply a mass-balance model that predicts the fraction of individuals shedding HuNoV in their stool based on measured concentrations in wastewater solids.

Methods: We measured HuNoV GII RNA in matched wastewater solids and liquid influent in 7 samples from a WWTP. We also applied the HuNoV GII assay to measure viral RNA in over 6000 wastewater solids samples from 145 WWTPs from across the United States daily to three times per week for up to five months. Measurements were made using digital droplet RT-PCR.

Results: HuNoV GII RNA preferentially adsorbs to wastewater solids where it is present at 1000 times the concentration in influent. Concentrations of HuNoV GII RNA correlate positively with clinical HuNoV positivity rates. Model output of the fraction of individuals shedding HuNoV is variable and uncertain, but consistent with indirect estimates of symptomatic HuNoV infections in the United States.

Impact statement: Illness caused by HuNoV is not reportable in the United States so there is limited data on disease occurrence. Wastewater monitoring can provide information about the community spread of HuNoV. Data from wastewater can be available within 24 h of sample receipt at a laboratory. Wastewater is agnostic to whether individuals seek medical care, are symptomatic, and the severity of illness. Knowledge gleaned from wastewater may be used by public health professionals to make recommendations on hand washing, surface disinfection, or other behaviors to reduce transmission of HuNoV, or medical doctors to inform clinical decision making.

Keywords: Disease; Environmental Monitoring; Epidemiology.

Fig. 1. Concentrations of HuNoV GII and PMMoV RNA in influent and solids samples collected on the same day over seven consecutive days between 28 February 2023 and 6 March 2023.

Each marker on the plot has an error bar, but the error bar is so small, it is covered by the marker. The error on the measurements is approximately 2–3%.

Fig. 2. HuNoV clinical positivity rate and concentrations in wastewater solids.

Top panels: Positivity rate for clinical specimens processed by the clinical laboratory (left) and by the sentinel laboratories participating in NREVSS (right). Middle panels and bottom panel: Concentrations of HuNoV GII RNA and HuNoV GII RNA normalized by PMMoV RNA, respectively. For the SJ WWTP, errors are provided as standard deviations as reported by the analysis software that incorporate variability among 10 replicates and Poisson error; if error bars are not visible, it is because they are smaller than the size of the marker. The error on the ratio is propagated from those of the values in the numerator and denominator. The 5-d trimmed average is shown as well as raw data as a solid line. For the national WWTP, raw data are shown for every WWTP, broken axes are needed to show higher measurements. The population-weighted average lines are shown as solid lines.

Fig. 3. Modeled F_shed for SJ WWTP an the national WWTPs.

Solid line is the median model output, and the 25th and 75th percentile model outputs are shown as dotted lines.