Assessment of biomarkers for normalization of SARS-CoV-2 concentrations in wastewater
Authors:   , Osman Aaliyah 
Published: February 2024

During the COVID-19 pandemic, the measurement of SARS-CoV-2 RNA levels in wastewater quickly emerged as an additional tool for monitoring and to provide an early warning system. This led to development of several regional, national and international projects aimed at applying this approach. The main principle is based on the detection of the viral signature in untreated wastewater to provide an indication of infection levels within connected populations. However, the concentration of the viral signature in wastewater can be impacted by dilution factors or population changes in the sewer shed, leading to misinterpretation of measurement results. Therefore, there is the need for normalization of wastewater to ensure accurate representation of infection numbers. The aim of this study was to evaluate different viral and bacterial markers in wastewater for their efficiency in normalizing SARS-CoV-2 WBE data, which will enhance the accuracy when interpreting the SARS-CoV-2 RNA concentrations in wastewater. Weekly sampling was conducted from two wastewater treatment plants (WWTP A and WWTP B) within the eThekwini district over a period of three months (July-October 2022). Three biomarkers (crAssphage, Bacteroides (HF 183), and Pepper Mild Motile Virus) where chosen for this study to ascertain the most suitable for WBE data normalization. Biomarker and SARS CoV-2 concentrations in the wastewater samples were determined using the droplet digital PCR (ddPCR). Physicochemical characteristics of the wastewater samples were also determined to identify the potential impact of these characteristics on the concentration of SARS-CoV-2 and the biomarkers. To determine the most suitable biomarker, correlation analysis and the Adaptive neuro fuzzy inference system (ANFIS) model was used. Average concentrations of SARS-CoV-2 in the sampled WWTPs ranged from 0.28 copies/µL to 9.57 copies/µL. Among the three biomarkers studied, crAssphage recorded the highest concentration compared to PMMoV and Bacteroides HF183 in both the WWTPs. CrAssphage recorded the highest concentration of 7943 (±7.07) copies/µL for WWTP A and 8006 (±4.24) copies/µL for WWTP B. The Bacteroides HF183 highest concentrations were 10116 (±120.91) copies/µL for WWTP A and 2474 (±117.37) copies/µL for WWTP B. PMMoV had concentrations of 46 (±4.24) copies/µL for WWTP A and 84,1 (±5.48) copies/µL for WWTP B. PMMoV concentrations were observed to be the highest at Week 1. CrAssphage showed a greater association during the trend analysis with SARS-CoV-2 (0.499) than the other two biomarkers for WWTP A, (HF 183 and SARS-CoV-2 (-0.191) and PMMoV and SARS-CoV 2 (-0.562)). Among the physicochemical factors studied, electrical conductivity and temperature had a significant correlation with SARS-CoV-2 and the crAssphage biomarker for both WWTPs. Using the ANFIS model, it was shown that the levels of the measured biomarker concentrations in wastewater had a significant association with chemical oxygen demand (COD), dissolved oxygen (DO), and volatile solids (VS). These results indicate a possible impact of these parameters on the concentration of these biomarkers in the wastewater. Furthermore, the viral RNA quantities of SARS-CoV-2 in wastewater were demonstrated to be influenced by other parameters such as electrical conductivity, pH and temperature. This indicates a difference in the physicochemical parameters that influence both biomarkers and SARS-CoV-2. However, when all physicochemical parameters, biomarkers and SARS-CoV-2 were combined, it was determined that the best biomarker was crAssphage, with potential impact from COD and the VS. The results of this study highlight the significance of including wastewater characteristic in WBE studies for reliable and accurate results. As shown in this study, crAssphage can serve ix as a biomarker for efficient WBE for COVID-19 surveillance. In addition, it has been demonstrated that the detection and quantification of targets of concern, including SARS-CoV 2, may be enhanced when combined with wastewater characteristics, which may enhance the monitoring of COVID-19 infections.