Wastewater Based Epidemiology in Countries with Poor Wastewater Treatment - Epidemiological Indicator Function of SARS-CoV-2 RNA in Surface Waters

Wastewater-based epidemiology (WBE) surveillance of COVID-19 and other outbreaks in the future is a challenge for developing countries, as the majority of households are not connected to sewerage systems. In December 2019, SARS-CoV-2 RNA was detected in the Danube River at a site severely affected by wastewaters of Belgrade. Rivers are much more complex systems in comparison to wastewaters, and additional efforts are needed to address all the factors which might influence the adoption of WBE as an alternative of targeting raw wastewater. The major objective was to provide a more detailed insight in the potential of SARS-CoV-2 surveillance in Serbian surface waters for epidemiological purposes. Water samples were collected at 12 sites at Sava and Danube in Belgrade during the fourth COVID-19 wave in Serbia started in late February 2021. SARS-CoV-2 RNA was quantified using RT-qPCR with primer sets targeting nucleocapsid (N1 and N2) and envelope (E) protein genes. Microbiological (standard faecal indicator bacteria and microbial faecal source tracking markers), epidemiological, physico-chemical and hydro-morphological parameters were analyzed in parallel. From 44 samples SARS-CoV-2 RNA was detectable in 31 samples but only in 4 concentrations were above the level of quantification. The results indicated that surveillance of SARS-CoV-2 RNA in surface waters could be used as epidemiological early-warning tool in countries with poor wastewater treatment. The performance of the applied approach including advanced sampling site characterization could be further improved by adaptation of the methodology for processing higher volumes of samples and enrichment factors which should enable to realize quantitative instead of qualitative data needed for WBE.