Evaluation of low-cost SARS-CoV-2 RNA purification methods for viral quantification by RT-qPCR and next-generation sequencing analysis: Implications for wider wastewater-based epidemiology adoption.

Based Epidemiology (WBE) consists of quantifying biomarkers in sewerage systems to derive real-time information on the health and/or lifestyle of the contributing population. WBE usefulness was vastly demonstrated in the context of the COVID-19 pandemic. Many methods for SARS-CoV-2 RNA determination in wastewater were devised, which vary in cost, infrastructure requirements and sensitivity. For most developing countries, implementing WBE for viral outbreaks, such as that of SARS-CoV-2, proved challenging due to budget, reagent availability and infrastructure constraints. In this study, we assessed low-cost methods for SARS-CoV-2 RNA quantification by RT-qPCR, and performed variant identification by NGS in wastewater samples. Results showed that the effect of adjusting pH to 4 and/or adding MgCl2 (25 mM) was negligible when using the adsorption-elution method, as well as basal physicochemical parameters in the sample. In addition, results supported the standardized use of linear rather than plasmid DNA for a more accurate viral RT-qPCR estimation. The modified TRIzol-based purification method in this study yielded comparable RT-qPCR estimation to a column-based approach, but provided better NGS results, suggesting that column-based purification for viral analysis should be revised. Overall, this work provides evaluation of a robust, sensitive and cost-effective method for SARS-CoV-2 RNA analysis that could be implemented for other viruses, for a wider WEB adoption.

Spatiotemporal Surveillance of SARS-CoV-2 in the Sewage of Three Major Urban Areas in Peru: Generating Valuable Data Where Clinical Testing Is Extremely Limited.

Peru has been severely affected by the COVID-19 pandemic. By January 2022, Peru had surpassed 200 000 COVID-19 deaths, constituting the highest death rate per capita worldwide. Peru has had several limitations during the pandemic: insufficient testing access, limited contact tracing, a strained medical infrastructure, and many economic hurdles. These limitations hindered the gathering of accurate information about infected individuals with spatial resolution in real time, a critical aspect of effectively controlling the pandemic. Wastewater monitoring for SARS-CoV-2 RNA offered a promising alternative for providing needed population-wide information to complement health care indicators. In this study, we demonstrate the feasibility and value of implementing a decentralized SARS-CoV-2 RNA wastewater monitoring system to assess the spatiotemporal distribution of COVID-19 in three major cities in Peru: Lima, Callao, and Arequipa. Our data on viral loads showed the same trends as health indicators such as incidence and mortality. Furthermore, we were able to identify hot spots of contagion within the surveyed urban areas to guide the efforts of health authorities. Viral decay in the sewage network of the cities studied was found to be negligible (<2%). Overall, our results support wastewater monitoring for SARS-CoV-2 as a valuable and cost-effective tool for monitoring the COVID-19 pandemic in the Peruvian context.