Abstract：
Wastewater-based SARS-CoV-2 epidemiology (WBE) has proven as an excellent tool to monitor pandemic dynamics supporting individual testing strategies. WBE can also be used as an early warning system for monitoring the emergence of novel pathogens or viral variants. However, for a timely transmission of results, sophisticated sample logistics and analytics performed in decentralized laboratories close to the sampling sites are required. Since multiple decentralized laboratories commonly use custom in-house workflows for sample purification and PCR-analysis, comparative quality control of the analytical procedures is essential to report reliable and comparable results. In this study, we performed an interlaboratory comparison at laboratories specialized for PCR and high-throughput-sequencing (HTS)-based WBE analysis. Frozen reserve samples from low COVID-19 incidence periods were spiked with different inactivated authentic SARS-CoV-2 variants in graduated concentrations and ratios. Samples were sent to the participating laboratories for analysis using laboratory specific methods and the reported viral genome copy numbers and the detection of viral variants were compared with the expected values. All PCR-laboratories reported SARS-CoV-2 genome copy equivalents (GCE) for all spiked samples with a mean intra- and inter-laboratory variability of 19 % and 104 %, respectively, largely reproducing the spike-in scheme. PCR-based genotyping was, in dependence of the underlying PCR-assay performance, able to predict the relative amount of variant specific substitutions even in samples with low spike-in amount. The identification of variants by HTS, however, required >100 copies/ml wastewater and had limited predictive value when analyzing at a genome coverage below 60 %. This interlaboratory test demonstrates that despite highly heterogeneous isolation and analysis procedures, overall SARS-CoV-2 GCE and mutations were determined accurately. Hence, decentralized SARS-CoV-2 wastewater monitoring is feasible to generate comparable analysis results. However, since not all assays detected the correct variant, prior evaluation of PCR and sequencing workflows as well as sustained quality control such as interlaboratory comparisons are mandatory for correct variant detection.
摘要：
基于废水的SARS-CoV-2流行病学（WBE）已被证明是监测大流行动态的出色工具，可支持个人测试策略。WBE也可用作监测新病原体或病毒变体出现的早期预警系统。然而,为了及时传达结果，需要在靠近采样点的分散实验室进行复杂的样品物流和分析。由于多个分散实验室通常使用定制的内部工作流程进行样品纯化和PCR分析，分析程序的比较质量控制对于报告可靠和可比的结果至关重要。在这项研究中,我们在专门用于PCR和基于高通量测序(HTS)的WBE分析的实验室进行了实验室间比较.来自低COVID-19发病率的冷冻储备样品以渐变的浓度和比例掺入不同的灭活的真正的SARS-CoV-2变体。使用实验室特异性方法将样品送到参与的实验室进行分析,并将报告的病毒基因组拷贝数和病毒变体的检测与预期值进行比较。所有PCR实验室都报告了所有加标样品的SARS-CoV-2基因组拷贝当量（GCE），平均实验室内和实验室间变异性分别为19％和104％，分别,在很大程度上再现了尖峰计划。基于PCR的基因分型是，依赖于潜在的PCR检测性能，能够预测变体特异性取代的相对量，甚至在具有低掺入量的样品中。通过HTS鉴定变体，然而,需要>100拷贝/mL的废水，并且在基因组覆盖率低于60％的情况下进行分析时，预测价值有限。该实验室间测试表明，尽管分离和分析程序高度异构，对SARS-CoV-2总体GCE和突变进行了准确测定.因此，分散的SARS-CoV-2废水监测是可行的，以产生可比的分析结果。然而,因为不是所有的检测都能检测到正确的变异，PCR和测序工作流程的事先评估以及持续的质量控制(如实验室间比较)对于正确的变异体检测是强制性的.
