https://ascelibrary.org/doi/10.1061/%28ASCE%29EE.1943-7870.0001994

Chik, A.H.S., Ho, J.J.Y., Srikanthan, N., Dhiyebi, H., Servos, M. (2022) Design and Validation of Sample Splitting Protocol for Comparison of SARS-CoV-2 Quantification in Wastewater. J Environ Eng (United States). 2022;148(8). https://ascelibrary.org/doi/10.1061/%28ASCE%29EE.1943-7870.0001994

Evaluations of analytical performance through interlaboratory comparisons and proficiency tests are underway globally for biomolecular-based methods [e.g., reverse-transcription quantitative polymerase chain reaction (RT-qPCR)] used in the surveillance of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater. These evaluations often rely on sharing a common reference wastewater sample that is split among participating laboratories. A known quantity of recovery surrogates can be introduced to the wastewater matrix by the coordinating laboratory as an exogenous control in a spike-and-recovery approach; however, split-sample comparisons are increasingly performed to evaluate in situ quantities of SARS-CoV-2 genetic signal native to the sample due to the lack of a universally accepted recovery surrogate of SARS-CoV-2. A reproducible procedure that minimizes the variability of SARS-CoV-2 genetic signal among split wastewater aliquots is therefore necessary to facilitate the method comparisons, especially when a large number of aliquots are required. Emerging literature has suggested that SARS-CoV-2 genetic signal in wastewater is linked to the solids fraction. Accordingly, a protocol that allows for equal distribution of solids content evenly among wastewater aliquots was also likely to facilitate even distribution of the SARS-CoV-2 genetic signal. Based on this premise, we reviewed existing sample splitting apparatus and approaches used for solids-based parameters in environmental samples. A portable batch reactor was designed, comprised of readily accessible materials and equipment. This design was validated through splitting of real wastewater samples collected from a municipal wastewater treatment facility serving a population with reported cases of COVID-19. This work applies well-established solid-liquid mixing theory and concepts that are likely unfamiliar to molecular microbiologists and laboratory analysts, providing (1) a prototype adaptable for a range of sample quantities, aliquot sizes, microbial targets, and water matrices; and (2) a pragmatic demonstration of critical considerations for design and validation of a reproducible and effective sample splitting protocol.

Highlights
• A novel wastewater-based risk index simplified understanding of viral load
• Significant correlation relationship between wastewater RNA loads and clinical data
• Thresholds derived from daily per capita viral loads and clinic Rt estimates
• Wastewater-based risk index valuable for decision-making of COVID-19 risk

See COVID-19 Dashboard: https://water.usask.ca/covid-19

https://ascelibrary.org/doi/10.1061/%28ASCE%29EE.1943-7870.0001994