Monitoring of SARS‐CoV‐2 concentration and circulation of variants of concern in wastewater of Leuven, Belgium

Abstract

Wastewater surveillance plays an important role in the management of the coronavirus disease 2019 (COVID‐19) pandemic all over the world. Using different wastewater collection points in Leuven, we wanted to investigate the use of wastewater surveillance as an early warning system for an uprise of infections and as a tool to follow the circulation of specific variants of concern (VOCs) in particular geographic areas. Wastewater samples were collected from local neighborhood sewers and from a large regional wastewater treatment plant (WWTP) in the area of Leuven, Belgium. After virus concentration, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) RNA was quantified by real‐time quantitative polymerase chain reaction (RT‐qPCR) and normalized with the human fecal indicator pepper mild mottle virus (PMMoV). A combination of multiplex RT‐qPCR assays was used to detect signature mutations of circulating VOCs. Fecal virus shedding of SARS‐CoV‐2 variants was measured in feces samples of hospitalized patients. In two residential sampling sites, a rise in wastewater SARS‐CoV‐2 concentration preceded peaks in positive cases. In the WWTP, viral load peaks were seen concomitant with the consecutive waves of positive cases caused by the original Wuhan SARS‐CoV‐2 strain and subsequent VOCs. During the Omicron BA.1 wave, the wastewater viral load increased to a lesser degree, even after normalization of SARS‐CoV‐2 concentration using PMMoV. This might be attributable to a lower level of fecal excretion of this variant. Circulation of SARS‐CoV‐2 VOCs Alpha, Delta, Omicron BA1/BA.2, and BA.4/BA.5 could be detected based on the presence of specific key mutations. The shift in variants was noticeable in the wastewater, with key mutations of two different variants being present simultaneously during the transition period. Wastewater‐based surveillance is a sensitive tool to monitor SARS‐CoV‐2 circulation levels and VOCs in larger regions. In times of reduced test capacity, this can prove to be highly valuable. Differences in excretion levels of various SARS‐CoV‐2 variants should however be taken into account when using wastewater surveillance to monitor SARS‐CoV‐2 circulation levels in the population.

Detection of measles virus genotype D8 in wastewater of Brussels capital region, Belgium, March 2024

“…Copies per µL extracted RNA isolated from the concentrated sewage samples correspond to copies per ml of untreated wastewater. Human faecal indicator pepper mild mottle virus (PMMoV) was quantified to normalize for differences in human waste input and changes in wastewater dilution (5).…”

“…Wastewater samples were obtained from 3 large regional WWTP. Since December 2020, we examine wastewater samples of the plant of Aquafin Leuven on a weekly basis to track circulation levels of SARS-CoV-2 (5). This plant drains wastewater from 8 municipalities in the region of Flemish Brabant, comprising of approximately 120,000 inhabitants.…”

“…In these WWTPs, samples of 24 hours influent wastewater were obtained with a time proportional sampler. Samples were stored at 4°C, and after transportation to the lab pretreatment and extraction of viral nucleic acids was performed as previously described (5).…”

Used paper tissues for pathogen identification in acute respiratory infection

“…Since the current study was not focused on immunocompromised populations, these parameters were not included in our analysis. An additional SARS‐CoV‐2 specific RT‐qPCR was carried out on samples positive for HCoV‐SARS using the 2019‐nCoV CDC EUA kit N1 primer‐probe set 5 …”

“…An additional SARS-CoV-2 specific RT-qPCR was carried out on samples positive for HCoV-SARS using the 2019-nCoV CDC EUA kit N1 primer-probe set. 5 Typing of respiratory pathogens from UPT based on partial sequencing was done as described previously for EV/RV, 6 SARS-CoV-2, 7 and influenza 8,9 . Complete genome sequencing of SARS-CoV-2 on RNA extracted from UPT was done with the nanopore technique using the ARTIC protocol as described in Wawina-Bokalanga et al 10 Ag-RDTs were carried out as described in the test instruction manual.…”