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
Applied Virology Laboratory, Department of Microbiology, Immunology and Parasitology, Federal University of Santa Catarina, Florianópolis, SC, Brazil.
Protozoology Laboratory, Department of Microbiology, Imunology and Parasitology, Federal University of Santa Catarina, Florianópolis, SC, Brazil.
Sci Total Environ. 2021 Jul 15;778:146198. doi: 10.1016/j.scitotenv.2021.146198. Epub 2021 Mar 8.
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 SARS-CoV-2 genome copies (GC) L), 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 SARS-CoV-2 GC L, ranging from 5.49 ± 0.02 log GC L (27th November 2019) to 6.68 ± 0.02 log GC L (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).
从2019年10月至2020年3月，对来自弗洛里亚诺波利斯（巴西圣卡塔琳娜州）的生活污水进行了严重急性呼吸综合征冠状病毒2（SARS-CoV-2）分析。取25毫升污水样本进行澄清处理，采用甘氨酸缓冲液法结合聚乙二醇沉淀法浓缩病毒，并用商用试剂盒提取病毒RNA。使用靶向N1、S和两个RNA依赖的RNA聚合酶（RdRp）区域的寡核苷酸通过逆转录定量聚合酶链反应（RT-qPCR）检测SARS-CoV-2 RNA。所有阳性样本的结果在独立实验室中通过不同的RT-qPCR系统进一步确认。对S和RdRp扩增子进行测序以确认与SARS-CoV-2的一致性。采用两种策略进行基因组测序；一种是不依赖序列的单引物扩增（SISPA）方法，另一种是使用Illumina的下一代测序（NGS）通过直接宏基因组学方法。2019年11月27日检测到SARS-CoV-2 RNA（5.49±0.02 log SARS-CoV-2基因组拷贝数（GC）/升），该检测结果经独立实验室和基因组测序分析确认。随后三次检测事件中的样本通过所有RT-qPCR检测均为阳性；这些阳性结果也经独立实验室确认。平均病毒载量为5.83±0.12 log SARS-CoV-2 GC/升，范围从5.49±0.02 log GC/升（2019年11月27日）到6.68±0.02 log GC/升（2020年3月4日）。我们的研究结果表明，自2019年11月以来，SARS-CoV-2可能已在巴西社区中未被检测到地传播，早于美洲报告的首例病例（2020年1月21日）。
