Wilhelm Alexander, Schoth Jens, Meinert-Berning Christina, Bastian Daniel, Blum Helmut, Elsinga Goffe, Graf Alexander, Heijnen Leo, Ho Johannes, Kluge Mariana, Krebs Stefan, Stange Claudia, Uchaikina Anna, Dolny Regina, Wurzbacher Christian, Drewes Jörg E, Medema Gertjan, Tiehm Andreas, Ciesek Sandra, Teichgräber Burkhard, Wintgens Thomas, Weber Frank-Andreas, Widera Marek
Goethe University Frankfurt, University Hospital, Institute for Medical Virology, Paul-Ehrlich-Str. 40, D-60596 Frankfurt, Germany.
Emschergenossenschaft/Lippeverband, Kronprinzenstraße 24, D-45128 Essen, Germany.
Sci Total Environ. 2023 Dec 10;903:166540. doi: 10.1016/j.scitotenv.2023.166540. Epub 2023 Aug 25.
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.
基于废水的严重急性呼吸综合征冠状病毒2型流行病学（WBE）已被证明是监测疫情动态、支持个体检测策略的优秀工具。WBE还可作为监测新型病原体或病毒变体出现的早期预警系统。然而，为了及时传输结果，需要在靠近采样点的分散实验室中进行复杂的样本物流和分析。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本纯化和PCR分析，因此分析程序的比较质量控制对于报告可靠且可比的结果至关重要。在本研究中，我们在专门从事基于PCR和高通量测序（HTS）的WBE分析的实验室之间进行了实验室间比较。将低新冠发病率时期的冷冻储备样本与不同浓度和比例的灭活真实严重急性呼吸综合征冠状病毒2型变体混合。样本被送往参与实验室，使用实验室特定方法进行分析，并将报告的病毒基因组拷贝数和病毒变体检测结果与预期值进行比较。所有PCR实验室都报告了所有加标样本的严重急性呼吸综合征冠状病毒2型基因组拷贝当量（GCE），实验室内部和实验室间的平均变异率分别为19%和104%，很大程度上重现了加标方案。基于PCR的基因分型能够根据基础PCR检测性能预测变体特异性替代的相对数量，即使在加标量较低的样本中也是如此。然而，通过HTS鉴定变体需要废水中>100拷贝/ml，并且在基因组覆盖率低于60%时分析的预测价值有限。这项实验室间测试表明，尽管分离和分析程序高度异质，但总体上严重急性呼吸综合征冠状病毒2型GCE和突变被准确测定。因此，分散的严重急性呼吸综合征冠状病毒2型废水监测对于生成可比的分析结果是可行的。然而，由于并非所有检测都能检测到正确的变体，因此必须对PCR和测序工作流程进行预先评估以及进行持续的质量控制，如实验室间比较，以正确检测变体。
