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

Abstract

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 log₁₀ 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 log₁₀ SARS-CoV-2 GC L^-1, ranging from 5.49 ± 0.02 log₁₀ GC L^-1 (27th November 2019) to 6.68 ± 0.02 log₁₀ 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).

Keywords: Brazil; COVID-19; Epidemiology; Human sewage; SARS-CoV-2; Surveillance.

Graphical abstract

Fig. 1

Evolution of the presence of SARS-CoV-2 RNA in urban sewage, Florianopolis, Brazil. Red dots represent negative results. Black dots represent log₁₀ SARS-CoV-2 genome copies per liter of urban sewage (M ± SE) from independent duplicate estimates. N1 RT-qPCR assay was used for quantification of SARS-CoV-2 genome copies per liter. All negative RT-qPCR and sample process controls in each sampling event were negative. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Supplementary Material Fig. 1

Maps of the sampling location in Florianópolis city, Santa Catarina State, Brazil.

Supplementary Material Fig. 2

Mapping of sequences from the SARS-CoV-2 positive sample (27th November 2019) with SARS-CoV-2 reference genome. A total of 95 reads were distributed along to the complete genome of SARS-CoV-2, corresponding to 25% of genome coverage.