Upstream SARS-CoV-2 variant surveillance at Breede Valley municipality using wastewater-based epidemiology

The COVID-19 pandemic has strained economies and health systems worldwide, particularly in low- and middle-income countries, where limited resources were further stretched by the crisis. In response, innovative strategies like wastewater-based epidemiology (WBE) have emerged as cost-effective and efficient alternatives to traditional surveillance. In Breede Valley Municipality, Western Cape, South Africa, weekly monitoring of wastewater treatment plants (WWTPs) showed consistently high SARS-CoV-2 viral loads in Rawsonville, especially of the Delta variant (AY.32 and B.1.617.2), despite Omicron's dominance in other regions. Total RNAs of all samples were extracted using the Qiagen RNeasy PowerSoil Kit, from which both RT-qPCR and whole genome sequencing were performed to determine the amount of SARS-CoV-2 RNA and their variants, respectively. Rawsonville had the highest median viral load, measured in genome copies per millilitre (gc/mL) at 2211 gc/mL, compared to Worcester (1790 gc/mL), De Doorns (1438 gc/mL), and Touws River (1253 gc/mL). Neighbourhoods in Rawsonville (RV) upstream of the main WWTP, where manholes were sampled, indicated that the suburban manhole on Grey Street (RV_G, 2347 gc/mL) and the peri-urban manhole on Rugby Street (RV_R, 714 gc/mL) were the primary contributors to the SARS-CoV-2 viral load. In contrast, the manhole near public toilets (RV_P, 470 gc/mL) had the lowest viral load. Notably, sequencing data indicated that only Omicron (BA.5) was detected in upstream sites RV_G and RV_R when Deltacron was present in Rawsonville, suggesting complex transmission dynamics requiring further study. These findings underscore the importance of targeted upstream surveillance to trace variant emergence and inform public health interventions.