The presence of SARS-CoV-2 RNA in human sewage in Santa Catarina, Brazil, November 2019

Fongaro, Gislaine; Stoco, Patrícia Hermes; Souza, Doris Sobral Marques; Grisard, Edmundo Carlos; Magri, Maria Elisa; Rogovski, Paula; Schörner, Marcos André; Barazzetti, Fernando Hartmann; Christoff, Ana Paula; de Oliveira, Luiz Felipe Valter; Bazzo, Maria Luiza; Wagner, Glauber; Hernández, Marta; Rodríguez-Lázaro, David

Human sewage from Florianopolis (Santa Catarina, Brazil) was analyzed for severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) from October 2019 until March 2020. Twenty five ml of sewage samples were clarified and viruses concentrated using a glycine buffer method coupled with polyethylene glycol precipitation, and viral RNA extracted using a commercial kit. SARS-CoV-2 RNA was detected by RT-qPCR using oligonucleotides targeting N1, S and two RdRp regions. The results of all positive samples were further confirmed by a different RT-qPCR system in an independent laboratory. S and RdRp amplicons were sequenced to confirm identity with SARS-CoV-2. Genome sequencing was performed using two strategies; a sequence-independent single-primer amplification (SISPA) approach, and by direct metagenomics using Illumina's NGS. SARS-CoV-2 RNA was detected on 27th November 2019 (5.49 ± 0.02 log(10) SARS-CoV-2 genome copies (GC) L(−1)), detection being confirmed by an independent laboratory and genome sequencing analysis. The samples in the subsequent three events were positive by all RT-qPCR assays; these positive results were also confirmed by an independent laboratory. The average load was 5.83 ± 0.12 log(10) SARS-CoV-2 GC L(−1), ranging from 5.49 ± 0.02 log(10) GC L(−1) (27th November 2019) to 6.68 ± 0.02 log(10) GC L(−1) (4th March 2020). Our findings demonstrate that SARS-CoV-2 was likely circulating undetected in the community in Brazil since November 2019, earlier than the first reported case in the Americas (21st January 2020).