Wastewater-based environmental surveillance enables the monitoring of SARS-CoV-2 dynamics within populations, offering critical epidemiological insights. Numerous workflows for tracking SARS-CoV-2 have been developed globally, underscoring the need for interlaboratory comparisons to ensure data consistency and comparability. An inter-calibration test was conducted among laboratories within the network monitoring SARS-CoV-2 in wastewater samples across the Lombardy region (Italy). The test aimed to evaluate data reliability and identify potential sources of variability using robust statistical approaches. Three wastewater samples were analyzed in parallel by four laboratories using identical pre-analytical (PEG-8000-based centrifugation) and analytical processes (qPCR targeting N1/N3 and Orf-1ab). A two-way ANOVA framework within Generalized Linear Models was applied, and multiple pairwise comparisons among laboratories were performed using the Bonferroni post hoc test. The statistical analysis revealed that the primary source of variability in the results was associated with the analytical phase. This variability was likely influenced by differences in the standard curves used by the laboratories to quantify SARS-CoV-2 concentrations, as well as the size of the wastewater treatment plants. The findings of this study highlight the importance of interlaboratory testing in verifying the consistency of analytical determinations and in identifying the key sources of variation.

Wastewater-based epidemiology has emerged as a complementary tool for the monitoring of COVID-19 pandemic waves and for the circulation of viral variants. The selection, standardization, and dynamics of different SARS-CoV-2 RNA targets in wastewater requires further investigation. In the present study, 106 wastewater samples were collected over a 24-month period from the wastewater treatment plant of Sondrio, north Italy, and were analyzed for the presence of SARS-CoV-2 RNA through the quantification of ORF1b, N1, and N3 gene targets via one-step real-time qPCR. In general, the three RNA targets demonstrated different performances and dynamics over the studied time period, underlying the usefulness of multiple viral targets in the surveillance of SARS-CoV-2 in wastewater. During the first 12 months, the quantification of the selected SARS-CoV-2 viral targets also correlated with the reported clinical cases in the same geographical area; however, from the overall data analysis this did not appear to significantly anticipate the epidemic waves. In conclusion, this study further supports the use of wastewater surveillance as a real time indicator of the human circulation of SARS-CoV-2. Moreover, the use of multiple viral gene targets has been shown to improve the reliability of SARS-CoV-2 surveillance in wastewater over time.