Association Between SARS-CoV-2 Viral Load in Wastewater and Reported Cases, Hospitalizations, and Vaccinations in Milan, March 2020 to November 2021

Abstract

This study evaluates the association between SARS-CoV-2 load in urban wastewater and surveillance indicators of infection prevalence and severity in Milan, Italy.

Several studies have demonstrated that wastewater surveillance can be used to monitor SARS-CoV-2 incidence.^1,2,3 This surveillance intends to overcome the limitations of traditional surveillance indicators,⁴ such as the number of positive tests, which depends on test availability and indications, or COVID-19–related hospitalizations, which occur weeks after the spread of SARS-CoV-2 and do not include mild or asymptomatic cases. This study evaluated the association between SARS-CoV-2 load in urban wastewater and surveillance indicators of infection prevalence and severity in Milan, Italy.

Methods

Sewage samples were collected approximately once a week from March 2020 to November 2021 in the Nosedo wastewater treatment plant, serving about 50% of the Milan population. SARS-CoV-2 RNA was measured in wastewater by amplifying the nucleocapsid gene, and viral load was calculated correcting for daily wastewater flow and population (eMethods in the Supplement). Aggregate epidemiological data about Milan were supplied by the Lombardy Region and included daily numbers of SARS-CoV-2–positive cases, COVID-19 hospitalizations, and individuals completing the vaccination cycle (2 doses for 2-dose vaccines or 1 for Ad26.COV2.S [Janssen/Johnson & Johnson]) by age group and sex. According to Italian legislation, informed consent and ethics committee approval were not required because the analyzed data were anonymous.

The SARS-CoV-2 load in wastewater was graphically compared with surveillance indicators of infection prevalence. Local polynomial regression was conducted and 95% confidence intervals calculated to assess trends in SARS-CoV-2 loads. Positive cases and COVID-19 hospitalizations in Milan were used to estimate the prevalence of infection and severe infection, assuming a 15-day viral excretion for each positive or hospitalized individual (eMethods in the Supplement). The daily proportion of vaccinated individuals was computed and standardized by age and sex to the population of patients with COVID-19 hospitalized before the vaccination campaign started. Such standardization was performed to measure the coverage of individuals at higher risk of hospitalization after SARS-CoV-2 infection. Analyses were performed with R version 4.0.2.

Results

Figure 1 presents positive cases and hospitalizations over the study period. Figure 2 presents wastewater SARS-CoV-2 loads. The vaccination campaign began in January 2021 and coverage progressively increased, reaching 75% (>85% for individuals at increased risk of hospitalization) in November 2021. The curves for wastewater load and hospitalized patients are similar until the increase in vaccination coverage. The curves for wastewater load and positive cases also are similar except during the first wave, which was characterized by a shortage of tests. Curves for positive cases and hospitalizations diverge from the curve for wastewater load as vaccination coverage increased, with decreases in cases and hospitalizations and increases in wastewater viral load. On November 30, 2021, despite the limited number of positive cases (n = 4672) and hospitalizations (n = 252), the wastewater load was 7.25 × 10⁹copies/d/1000 people (95% CI, 2.43-24.80 × 10⁹), comparable with values observed during the second wave (November 10, 2020; 12.30 × 10⁹copies/d/1000 people, 95% CI, 4.71-22.31 × 10⁹), before the vaccination campaign started.

Figure 1. SARS-CoV-2–Positive Cases and Patients Hospitalized With COVID-19.

A, Estimate of the daily number of positive cases and B, hospitalized patients actively shedding SARS-CoV-2, based on the data of new positives and newly hospitalized patients in Milan, Italy.

Figure 2. SARS-CoV-2 Load in Urban Wastewater.

SARS-CoV-2 load in wastewater (individual points) in Milan, Italy, and daily estimate with 95% CIs (smoothed line and shaded area).

Discussion

In Milan, high wastewater SARS-CoV-2 loads were found when vaccination coverage was high and traditional surveillance indicators suggested limited SARS-CoV-2 prevalence. This result suggests that there was significant circulating virus in the population during this period, including among vaccinated individuals. The SARS-CoV-2 circulation among vaccinated individuals may create modest evolutionary pressure toward resistance to the host’s immune response, making variants with significant transmission advantages more competitive. The current spread of the Omicron variant supports this theory.⁵

This study is limited by the difficulty in translating SARS-CoV-2 wastewater loads into infection prevalence because the variability of loads is affected by factors that can be controlled only partially.⁴ Nonetheless, the magnitude of the observed trends supports the value of wastewater surveillance to monitor the spread of SARS-CoV-2. In addition, the study was limited to a single city.

The results suggest that vaccines are effective in protecting against symptomatic and severe disease, but that, with high vaccination rates, standard surveillance metrics may not accurately estimate the spread of SARS-CoV-2. Thus, wastewater surveillance may be important as an early warning of virus circulation. These results strengthen the scientific basis of the recommendations from the Centers for Disease Control and Prevention National Wastewater Surveillance System and European Commission to establish systematic SARS-CoV-2 wastewater surveillance networks.

Section Editors: Jody W. Zylke, MD, Deputy Editor; Kristin Walter, MD, Associate Editor.

Supplement.

eMethods