Diagnostic Laboratory, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 11 Stavrou Voutyra str., 54627, Thessaloniki, Greece.
Department of Hygiene and Epidemiology, Faculty of Medicine, University of Thessaly, 22 Papakyriazi str., 41222 Larissa, Greece.
Sci Total Environ. 2022 Oct 20;844:156932. doi: 10.1016/j.scitotenv.2022.156932. Epub 2022 Jun 24.
Conventional SARS-CoV-2 surveillance based on genotyping of clinical samples is characterized by challenges related to the available sequencing capacity, population sampling methodologies, and is time, labor, and resource-demanding. Wastewater-based variant surveillance constitutes a valuable supplementary practice, since it does not require extensive sampling, and provides information on virus prevalence in a timely and cost-effective manner. Consequently, we developed a sensitive real-time RT-PCR-based approach that exclusively amplifies and quantifies SARS-CoV-2 genomic regions carrying the S:Δ69/70 deletion, indicative of the Omicron BA.1 variant, in wastewater. The method was incorporated in the analysis of composite daily samples taken from the main Wastewater Treatment Plant of Thessaloniki, Greece, from 1 December 2021. The applicability of the methodology is dependent on the epidemiological situation. During Omicron BA.1 global emergence, Thessaloniki was experiencing a massive epidemic wave attributed solely to the Delta variant, according to genomic surveillance data. Since Delta does not possess the S:Δ69/70, the emergence of Omicron BA.1 could be monitored via the described methodology. Omicron BA.1 was detected in sewage samples on 19 December 2021 and a rapid increase of its viral load was observed in the following 10-day period, with an estimated early doubling time of 1.86 days. The proportion of the total SARS-CoV-2 load attributed to BA.1 reached 91.09 % on 7 January, revealing a fast Delta-to-Omicron transition pattern. The detection of Omicron BA.1 subclade in wastewater preceded the outburst of reported (presumable) Omicron cases in the city by approximately 7 days. The proposed wastewater surveillance approach based on selective PCR amplification of a genomic region carrying a deletion signature enabled rapid, real-time data acquisition on Omicron BA.1 prevalence and dynamics during the slow remission of the Delta wave. Timely provision of these results to State authorities readily influences the decision-making process for targeted public health interventions, including control measures, awareness, and preparedness.
基于临床样本基因分型的传统 SARS-CoV-2 监测具有以下挑战：测序能力有限、人群采样方法、时间、劳动力和资源需求。污水变异监测构成了一种有价值的补充实践，因为它不需要广泛的采样，并且能够及时、经济高效地提供病毒流行情况的信息。因此，我们开发了一种敏感的基于实时 RT-PCR 的方法，该方法专门扩增和定量污水中携带 S:Δ69/70 缺失的 SARS-CoV-2 基因组区域，该缺失表明是 Omicron BA.1 变体。该方法被纳入对希腊塞萨洛尼基主要污水处理厂从 2021 年 12 月 1 日起每天采集的复合样本的分析中。该方法的适用性取决于流行病学情况。在 Omicron BA.1 全球出现期间，根据基因组监测数据，塞萨洛尼基正经历一场仅由 Delta 变体引起的大规模疫情。由于 Delta 变体不具有 S:Δ69/70，因此可以通过描述的方法监测 Omicron BA.1 的出现。2021 年 12 月 19 日在污水样本中检测到 Omicron BA.1，随后在接下来的 10 天内观察到其病毒载量迅速增加，早期倍增时间估计为 1.86 天。1 月 7 日，BA.1 占总 SARS-CoV-2 负荷的比例达到 91.09%，显示出快速的 Delta-Omicron 转换模式。污水中 Omicron BA.1 亚系的检测比该市报告（推测的）Omicron 病例的爆发提前了大约 7 天。基于选择性 PCR 扩增携带缺失特征的基因组区域的污水监测方法，能够快速实时获取 Omicron BA.1 流行率和动态数据，同时缓慢缓解 Delta 浪潮。及时向国家当局提供这些结果，有助于影响针对目标公共卫生干预措施（包括控制措施、意识和准备）的决策过程。
