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 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).
Keywords: Brazil; COVID-19; Epidemiology; Human sewage; SARS-CoV-2; Surveillance.
【저자키워드】 COVID-19, Brazil, SARS-CoV-2, Epidemiology, Surveillance, Human sewage, 【초록키워드】 Brazil, SARS-CoV2, Human, Sequencing, NGS, severe acute respiratory syndrome Coronavirus, virus, Laboratory, Severe acute respiratory syndrome, RT-qPCR, Genome sequencing, SARS-CoV-2 genome, Community, RdRP, SARS-CoV-2 RNA, Viral RNA, Illumina, Coronavirus-2, Analysis, Polyethylene glycol, Amplicon, glycine, glycol, identity, acute respiratory syndrome, acute respiratory syndrome coronavirus, positive result, average, acute respiratory syndrome coronavirus-2, sequence, circulating, oligonucleotides, America, positive, oligonucleotide, Americas, positive sample, buffer, regions, approach, FIVE, event, independent, log, analyzed, sequenced, reported, subsequent, was performed, single-primer amplification, viral RNA extracted,