Interlaboratory comparison using inactivated SARS-CoV-2 variants as a feasible tool for quality control in COVID-19 wastewater monitoring.

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.

Analyzing community wastewater in sub-sewersheds for the small-scale detection of SARS-CoV-2 variants in a German metropolitan area.

Wastewater surveillance of SARS-CoV-2 proved useful, including for identifying the local appearance of newly identified virus variants. Previous studies focused on wastewater treatment plants (WWTP) with sewersheds of several hundred thousand people or at single building level, representing only a small number of people. Both approaches may prove inadequate for small-scale intra-urban inferences for early detection of emerging or novel virus variants. Our study aims (i) to analyze SARS-CoV-2 single nucleotide variants (SNVs) in wastewater of sub-sewersheds and WWTP using whole genome sequencing in order to (ii) investigate the potential of small-scale detection of novel known SARS-CoV-2 variants of concern (VOC) within a metropolitan wastewater system. We selected three sub-sewershed sampling sites, based on estimated population- and built environment-related indicators, and the inlet of the receiving WWTP in the Ruhr region, Germany. Untreated wastewater was sampled weekly between October and December 2021, with a total of 22 samples collected. SARS-CoV-2 RNA was analyzed by RT-qPCR and whole genome sequencing. For all samples, genome sequences were obtained, while only 13 samples were positive for RT-qPCR. We identified multiple specific SARS-CoV-2 SNVs in the wastewater samples of the sub-sewersheds and the WWTP. Identified SNVs reflected the dominance of VOC Delta at the time of sampling. Interestingly, we could identify an Omicron-specific SNV in one sub-sewershed. A concurrent wastewater study sampling the same WWTP detected the VOC Omicron one week later. Our observations suggest that the small-scale approach may prove particularly useful for the detection and description of spatially confined emerging or existing virus variants circulating in populations. Future studies applying small-scale sampling strategies taking into account the specific features of the wastewater system will be useful to analyze temporal and spatial variance in more detail.

Early Detection of SARS-CoV-2 Omicron BA.4 and BA.5 in German Wastewater.

Wastewater-based SARS-CoV-2 epidemiology (WBE) has been established as an important tool to support individual testing strategies. The Omicron sub-variants BA.4/BA.5 have spread globally, displacing the preceding variants. Due to the severe transmissibility and immune escape potential of BA.4/BA.5, early monitoring was required to assess and implement countermeasures in time. In this study, we monitored the prevalence of SARS-CoV-2 BA.4/BA.5 at six municipal wastewater treatment plants (WWTPs) in the Federal State of North Rhine-Westphalia (NRW, Germany) in May and June 2022. Initially, L452R-specific primers/probes originally designed for SARS-CoV-2 Delta detection were validated using inactivated authentic viruses and evaluated for their suitability for detecting BA.4/BA.5. Subsequently, the assay was used for RT-qPCR analysis of RNA purified from wastewater obtained twice a week at six WWTPs. The occurrence of L452R carrying RNA was detected in early May 2022, and the presence of BA.4/BA.5 was confirmed by variant-specific single nucleotide polymorphism PCR (SNP-PCR) targeting E484A/F486V and NGS sequencing. Finally, the mutant fractions were quantitatively monitored by digital PCR, confirming BA.4/BA.5 as the majority variant by 5 June 2022. In conclusion, the successive workflow using RT-qPCR, variant-specific SNP-PCR, and RT-dPCR demonstrates the strength of WBE as a versatile tool to rapidly monitor variants spreading independently of individual test capacities.

Wastewater surveillance allows early detection of SARS-CoV-2 omicron in North Rhine-Westphalia, Germany.

Wastewater-based epidemiology (WBE) has demonstrated its importance to support SARS-CoV-2 epidemiology complementing individual testing strategies. Due to their immune-evasive potential and the resulting significance for public health, close monitoring of SARS-CoV-2 variants of concern (VoC) is required to evaluate the regulation of early local countermeasures. In this study, we demonstrate a rapid workflow for wastewater-based early detection and monitoring of the newly emerging SARS-CoV-2 VoCs Omicron in the end of 2021 at the municipal wastewater treatment plant (WWTP) Emschermuendung (KLEM) in the Federal State of North-Rhine-Westphalia (NRW, Germany). Initially, available primers detecting Omicron-related mutations were rapidly validated in a central laboratory. Subsequently, RT-qPCR analysis of purified SARS-CoV-2 RNA was performed in a decentral PCR laboratory in close proximity to KLEM. This decentralized approach enabled the early detection of K417N present in Omicron in samples collected on 8th December 2021 and the detection of further mutations (N501Y, Δ69/70) in subsequent biweekly sampling campaigns. The presence of Omicron in wastewater was confirmed by next generation sequencing (NGS) in a central laboratory with samples obtained on 14th December 2021. Moreover, the relative increase of the mutant fraction of Omicron was quantitatively monitored over time by dPCR in a central PCR laboratory starting on 12th December 2021 confirming Omicron as the dominant variant by the end of 2021. In conclusions, WBE plays a crucial role in surveillance of SARS-CoV-2 variants and is suitable as an early warning system to identify variant emergence. In particular, the successive workflow using RT-qPCR, RT-dPCR and NGS demonstrates the strength of WBE as a versatile tool to monitor variant spreading.

Performance evaluation and model-based optimization of the mainstream deammonification in an integrated fixed-film activated sludge reactor.

This study aimed to model and optimize mainstream deammonification in an integrated fixed-film activated sludge (IFAS) pilot plant under natural seasonal temperature variations. The effect of gradually decreasing temperature on the performance was evaluated during a winter season and a transition period to summer conditions, and the correlation of the performance parameters was investigated using principal component analysis (PCA). The optimization of intermittent aeration in the long-term (30 days) dynamic conditions with on/off ratio and dissolved oxygen (DO) set-point control was used to maximize the N-removal rate (NRR) and N-removal efficiency (NRE). Optimization results (DO set-point of 0.2-0.25 mgO2/L, and on/off ratio of 0.05) increased the NRE and NRR of total inorganic N (daily average) from 30% to > 50% and 15 gN/m3d to 25 gN/m3d, respectively. This novel long-term optimization strategy is a powerful tool for enhancing the efficiency in mainstream deammonification.

A module-based approach for elimination of organic micropollutants at wastewater treatment plants.

A technical feasibility study was carried out at the wastewater treatment plant (WWTP) Hamm-West in 2018, which included preliminary planning for the improvement of the plant, using different advanced wastewater technologies. The results of the technical feasibility study show that the application of activated carbon or ozone, in combination with an additional filtration system, can not only remove organic micropollutants efficiently but can also significantly improve the quality of other standard parameters in the WWTP effluent. This technical feasibility study, along with seven other studies, is part of the module-based approach the Emschergenossenschaft and Lippeverband (EGLV) is pursuing in order to improve wastewater treatment plants with advanced treatment systems. Finally, the module-based approach can be used to pair the most suitable WWTPs with the best applicable technologies to improve the treatment process in the whole Lippe catchment area.

Determination of chemical oxygen demand (COD) using an alternative wet chemical method free of mercury and dichromate.

Worldwide, the standard methods for the determination of the important wastewater parameter chemical oxygen demand (COD) are still based on the use of the hazardous chemicals, mercury sulfate and chromium(VI). However, due to their properties they are meanwhile classified as "priority pollutants" and shall be phased out or banned in the frame of REACH (current European Chemical Law: Registration, Evaluation, Authorization and restriction of Chemicals) by the European Union. Hence, a new wet-chemical method free of mercury and chromium(VI) was developed. Manganese(III) was used as oxidant and silver nitrate for the removal of chloride ions. The quantification was performed by back titration of manganese(III) with iron(II) as done in the standard method. In order to minimize losses of organic substances during the precipitation of silver chloride, suspended and colloid organic matter had to be separated by precipitation of aluminum hydroxide in a first step. In these cases, two fractions, one of the suspended and colloid matters and a second of the dissolved organic substances, are prepared and oxidized separately. The method was tested with potassium hydrogen phthalate (KHP) as conventional COD reference substance and different types of wastewater samples. The oxidation of KHP was reproducible in a COD range of 20-500 mg/L with a mean recovery rate of 88.7% in comparison to the standard COD method (DIN 38409-41). Also in presence of 1000 mg/L chloride a recovery rate of 84.1% was reached. For a series of industrial and municipal wastewater samples a high correlation (R2 = 0.9935) to the standard method with a mean recovery rate of 78.1% (±5.2%) was determined. Even though the results of the new method are not 100% of the standard method, its high correlation to the standard method and reproducibility offers an environmentally benign alternative method with no need to purchase new laboratory equipment.