Development of sensitive and high-throughput virus concentration methods for wastewater surveillance

Abstract

This thesis aimed to develop diverse virus concentration methods to obtain virus signals in wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) and influenza A/B viruses (IAV/IBV), and apply the established protocols to longitudinal monitoring for public health information. Firstly, an ultracentrifugation-based method was developed to achieve sensitive detection of SARS-CoV-2 in wastewater, based on a comprehensive evaluation of eleven virus concentration methods from different method principles and two RNA extraction methods. With this tool, a six-month longitudinal monitoring was performed across three wastewater treatment plants (WWTPs) during the 4th pandemic wave in Hong Kong and significant correlations were found between wastewater virus concentration and new cases number within the catchment areas. Additionally, normalization by pepper mild mottle virus (PMMoV) or flow rates did not enhance the correlations. Moreover, the SARS-CoV-2 viral load in wastewater effectively reflected the change of epidemiological scenarios in the community. This pilot study demonstrated the sensitivity and applicability of the established protocol to track the pandemic trends in the community. Secondly, a polyethylene glycol-based (PEG-based) method was developed to increase sample testing capacity for scaling up the wastewater surveillance programme, based on a systematic evaluation of different precipitation methods in the aspects of processing volumes, flocculants, extraction methods, centrifugation and incubation time lengths. By applying this high-throughput tool, a large-scale nine-month daily SARS-CoV-2 wastewater surveillance was conducted across twelve WWTPs during the 5th pandemic wave. The wastewater measurements tracked the pandemic peaks three days earlier than the reported cases number. Additionally, two methods were established to estimate prevalence/incidence rates from wastewater and revealed the underestimation of reported cases number when clinical testing was overwhelmed. Moreover, several public health metrics were derived from wastewater measurements to reflect the development of the pandemic for decision-making by stakeholders. This intensive large-scale study facilitated the interpretation of wastewater measurements into communicable and actionable public health information. Thirdly, two protocols were developed for detecting IAV/IBV virus types and subtypes in wastewater samples and were applied to the established SARS-CoV-2 wastewater surveillance network for an eight-month longitudinal monitoring. The city-wide IAV virus concentration in wastewater was associated with the positive rates and influenza-like illness plus rates (ILI+) of clinical respiratory specimens and increased significantly after the withdrawal of the mask mandate. Additionally, H1pdm was identified as the major circulation subtype in the community. This sentinel IAV/IBV work implied the efficacy of the established wastewater surveillance network for tracking infectious virus circulation patterns and controlling other pathogens during the post-pandemic era.