Optimization and Application of a Multiplex Digital PCR Assay for the Detection of SARS-CoV-2 Variants of Concern in Belgian Influent Wastewater.

Since the beginning of the COVID-19 pandemic, the wastewater-based epidemiology (WBE) of SARS-CoV-2 has been used as a complementary indicator to follow up on the trends in the COVID-19 spread in Belgium and in many other countries. To further develop the use of WBE, a multiplex digital polymerase chain reaction (dPCR) assay was optimized, validated and applied for the measurement of emerging SARS-CoV-2 variants of concern (VOC) in influent wastewater (IWW) samples. Key mutations were targeted in the different VOC strains, including SΔ69/70 deletion, N501Y, SΔ241 and SΔ157. The presented bioanalytical method was able to distinguish between SARS-CoV-2 RNA originating from the wild-type and B.1.1.7, B.1.351 and B.1.617.2 variants. The dPCR assay proved to be sensitive enough to detect low concentrations of SARS-CoV-2 RNA in IWW since the limit of detection of the different targets ranged between 0.3 and 2.9 copies/µL. This developed WBE approach was applied to IWW samples originating from different Belgian locations and was able to monitor spatio-temporal changes in the presence of targeted VOC strains in the investigated communities. The present dPCR assay developments were realized to bring added-value to the current national WBE of COVID-19 by also having the spatio-temporal proportions of the VoC in presence in the wastewaters.

Factors influencing SARS-CoV-2 RNA concentrations in wastewater up to the sampling stage: A systematic review.

Wastewater-based surveillance (WBS) for SARS-CoV-2 RNA is a promising complementary approach to monitor community viral circulation. A myriad of factors, however, can influence RNA concentrations in wastewater, impeding its epidemiological value. This article aims to provide an overview and discussion of factors up to the sampling stage that impact SARS-CoV-2 RNA concentration estimates in wastewater. To this end, a systematic review was performed in three databases (MEDLINE, Web of Science and Embase) and two preprint servers (MedRxiv and BioRxiv). Two authors independently screened and selected articles published between January 1, 2019 and May 4, 2021. A total of 22 eligible articles were included in this systematic review. The following factors up to sampling were identified to have an influence on SARS-CoV-2 RNA concentrations in wastewater and its interpretation: (i) shedding-related factors, including faecal shedding parameters (i.e. shedding pattern, recovery, rate, and load distribution), (ii) population size, (iii) in-sewer factors, including solid particles, organic load, travel time, flow rate, wastewater pH and temperature, and (iv) sampling strategy. In conclusion, factors influencing SARS-CoV-2 RNA concentration estimates in wastewater were identified and research gaps were discussed. The identification of these factors supports the need for further research on WBS for COVID-19.

An alternative approach for bioanalytical assay optimization for wastewater-based epidemiology of SARS-CoV-2.

Wastewater-based epidemiology of SARS-CoV-2 could play a role in monitoring the spread of the virus in the population and controlling possible outbreaks. However, sensitive sample preparation and detection methods are necessary to detect trace levels of SARS-CoV-2 RNA in influent wastewater (IWW). Unlike predecessors, method optimization of a SARS-CoV-2 RNA concentration and detection procedure was performed with IWW samples with high viral SARS-CoV-2 RNA loads. This is of importance since the SARS-CoV-2 genome in IWW might have already been subject to in-sewer degradation into smaller genome fragments or might be present in a different form (e.g. cell debris, …). Centricon Plus-70 (100 kDa) centrifugal filter devices resulted in the lowest and most reproducible Ct-values for SARS-CoV-2 RNA. Lowering the molecular weight cut-off did not improve our limit of detection and quantification (approximately 100 copies/µL for all genes). Quantitative polymerase chain reaction (qPCR) was employed for the amplification of the N1, N2, N3 and E-gene fragments. This is one of the first studies to apply digital polymerase chain reaction (dPCR) for the detection of SARS-CoV-2 RNA in IWW. dPCR showed high variability at low concentration levels (100 copies/µL), indicating that variability in bioanalytical methods for wastewater-based epidemiology of SARS-CoV-2 might be substantial. dPCR results in IWW were in line with the results found with qPCR. On average, the N2-gene fragment showed high in-sample stability in IWW for 10 days of storage at 4 °C. Between-sample variability was substantial due to the low native concentrations in IWW. Additionally, the E-gene fragment proved to be less stable compared to the N2-gene fragment and showed higher variability. Freezing the IWW samples resulted in a 10-fold decay of loads of the N2- and E-gene fragment in IWW.