Environmental Dissemination of SARS-CoV-2: An Analysis Employing Crassphage and Next-Generation Sequencing Protocols

This study aimed to investigate the dissemination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in water samples obtained during the coronavirus disease 2019 pandemic period, employing cross-assembly phage (crAssphage) as a fecal contamination biomarker and next-generation sequencing protocols to characterize SARS-CoV-2 variants. Raw wastewater and surface water (stream and sea) samples were collected for over a month in Rio de Janeiro, Brazil. Ultracentrifugation and negatively charged membrane filtration were employed for viral concentration of the wastewater and surface water samples, respectively. Viruses were detected and quantified by (RT-)qPCR applying TaqMan® system protocols. SARS-CoV-2 RNA signals were detected in 92.5% (37/40) of the wastewater samples and in 31.25% (10/32) of the stream water samples, but not in seawater samples. CrAssphage was detected in 100% of the wastewater samples, 93.75% (30/32) of the stream samples, and in 2/4 of the seawater samples. CrAssphage detection and high concentrations in stream surface waters (median 8.95 log₁₀ gc/L) revealed diffuse contamination by domestic wastewater in a region with high sanitary coverage. The correlations detected between SARS-CoV-2 data and the moving averages of clinical cases per capita over the sampling period were moderate to strong when applying a 13-day offset, regardless of normalization by crAssphage data or not. Sequencing of the receptor-binding domain of the spike protein confirmed the detection of SARS-CoV-2, but did not characterize the circulating variant. On the other hand, the whole genome sequencing protocol identified circulation of the Gamma variant, corroborating the sampling period clinical data.