Calibration of Methods for SARS-CoV-2 Environmental Surveillance: A Case Study from Northwest Tuscany

Abstract

The current pandemic has provided an opportunity to test wastewater-based epidemiology (WBE) as a complementary method to SARS-CoV-2 monitoring in the community. However, WBE infection estimates can be affected by uncertainty factors, such as heterogeneity in analytical procedure, wastewater volume, and population size. In this paper, raw sewage SARS-CoV-2 samples were collected from four wastewater treatment plants (WWTPs) in Tuscany (Northwest Italy) between February and December 2021. During the surveillance period, viral concentration was based on polyethylene glycol (PEG), but its precipitation method was modified from biphasic separation to centrifugation. Therefore, in parallel, the recovery efficiency of each method was evaluated at lab-scale, using two spiking viruses (human coronavirus 229E and mengovirus vMC0). SARS-CoV-2 genome was found in 80 (46.5%) of the 172 examined samples. Lab-scale experiments revealed that PEG precipitation using centrifugation had the best recovery efficiency (up to 30%). Viral SARS-CoV-2 load obtained from sewage data, adjusted by analytical method and normalized by population of each WWTP, showed a good association with the clinical data in the study area. This study highlights that environmental surveillance data need to be carefully analyzed before their use in the WBE, also considering the sensibility of the analytical methods.

Critical Needs for Integrated Surveillance: Wastewater-Based and Clinical Epidemiology in Evolving Scenarios with Lessons Learned from SARS-CoV-2

“… Method A: During the initial four months of this study (February 2021 to May 2021), the starting sample volume was 250 ml, and the recovery phase relied on biphasic separation using the WHO method for poliovirus environmental surveillance (WHO, 2003 ) adapted by the ISS (La Rosa et al, 2020 ). Method B: The protocol was updated in June 2021 (La Rosa et al, 2021b ) following the method of Wu et al ( 2020 ), in which 45 ml of sewage was analyzed and the recovery phase was performed through centrifugation, as described by Verani et al ( 2022 ). …”

“…Method B: The protocol was updated in June 2021 (La Rosa et al, 2021b ) following the method of Wu et al ( 2020 ), in which 45 ml of sewage was analyzed and the recovery phase was performed through centrifugation, as described by Verani et al ( 2022 ).…”

Wastewater-Based Epidemiology for Viral Surveillance from an Endemic Perspective: Evidence and Challenges

“…From October 2021 to September 2022, 197 weekly 24 h composite samples of raw wastewater were collected at the inlets of four urban wastewater treatment plants (WWTPs) in northwest Tuscany and were stored at 4 °C before analysis (within 48 h). The WWTPs chosen in this study were those involved in the surveillance network of the national project “Environmental Surveillance of SARS-CoV-2 by urban sewages in Italy” (SARI), operated according to an EU recommendation [ 25 ], as previously described [ 26 , 27 ]. Briefly, the studied WWTPs served populations of between 42,931 (WWTP1) and 110,871 (WWTP3), and there were some differences in the sewer network structures and population composition.…”

“…The extraction of viral nucleic acids was performed with the above-mentioned commercial kit, which allows the simultaneous extraction of DNA and RNA using magnetic silica beads, according to the manufacturer’s instructions [ 26 ]. Briefly, 2 mL of guanidine–thiocyanate-based lysis buffer was added to the pellet obtained after the concentration process.…”

“…Data on SARS-CoV-2 related to the same four WWTPs were extracted from a previous study [ 26 ] in order to cover three seasons in the 2021 period, namely, winter (20 data), spring (52 data), and summer (52 data). These data were compared with the monitoring data of the present study.…”