Reproducibility and sensitivity of 36 methods to quantify the SARS-CoV-2 genetic signal in raw wastewater: findings from an interlaboratory methods evaluation in the U.S.

In response to COVID-19, the international water community rapidly developed methods to quantify the SARS-CoV-2 genetic signal in untreated wastewater. Wastewater surveillance using such methods has the potential to complement clinical testing in assessing community health. This interlaboratory assessment evaluated the reproducibility and sensitivity of 36 standard operating procedures (SOPs), divided into eight method groups based on sample concentration approach and whether solids were removed. Two raw wastewater samples were collected in August 2020, amended with a matrix spike (betacoronavirus OC43), and distributed to 32 laboratories across the U.S. Replicate samples analyzed in accordance with the project's quality assurance plan showed high reproducibility across the 36 SOPs: 80% of the recovery-corrected results fell within a band of ±1.15 log10 genome copies per L with higher reproducibility observed within a single SOP (standard deviation of 0.13 log10). The inclusion of a solids removal step and the selection of a concentration method did not show a clear, systematic impact on the recovery-corrected results. Other methodological variations (e.g., pasteurization, primer set selection, and use of RT-qPCR or RT-dPCR platforms) generally resulted in small differences compared to other sources of variability. These findings suggest that a variety of methods are capable of producing reproducible results, though the same SOP or laboratory should be selected to track SARS-CoV-2 trends at a given facility. The methods showed a 7 log10 range of recovery efficiency and limit of detection highlighting the importance of recovery correction and the need to consider method sensitivity when selecting methods for wastewater surveillance.

5-Cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction in a mesophilic anaerobic digester: measuring redox behavior, differentiating abiotic reduction, and comparing FISH response as an activity indicator.

The tetrazolium salt 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) has been widely applied to assess microbiological activity in environmental samples. CTC reduction has previously been quantified in a variety of anaerobic systems (i.e., fermentative, nitrate reducing, sulfate reducing) using direct microscopy, solvent extraction, and flow cytometry. In this work, extracellular CTC reduction was observed and distinguished from its intercellular counterparts by the amorphous character and near uniform fluorescence of the resulting formazan precipitates (CTF). Fluorescence yielded by non-cellular-associated formazan precipitates bleached much more rapidly than CTF formed within cells under identical UV exposure (<2 min). Dehydrogenase activity assays and fluorescent in situ hybridization (FISH) were simultaneously carried out in microcosms containing active anaerobic digester biomass, propylene glycol, and settled sewage centrate for direct comparison. In substrate limited microcosms, quantitative FISH measurements remained well above their detection limit indicating sustained intercellular ribosomal RNA concentrations over a 5-day period, while dehydrogenase assays (CTC) decreased to background levels within 14 h of substrate limitation. Results from this work suggest that CTC reduction in cell-free samples may impede accurate enzyme activity measurements, particularly when quantification involves solvent extraction, flow cytometry, or software-aided counting. In addition, activity assessment in anaerobic digesters using FISH and CTC reduction assays may be comparable until substrate becomes limited.