Spiked Virus Level Needed To Correctly Assess Enteric Virus Recovery in Water Matrices

Abstract

Quantitative microbial risk assessment (QMRA) identifies human enteric viruses in municipal wastewater as the pathogen group requiring the highest log reductions for various reuse applications. However, the performance of methods for estimating virus concentration is not well understood, and without performance assessment, actual risks are likely severely underestimated. To evaluate the efficiency of virus recovery from water, a water sample is often spiked with “known” amounts of virus, and the virus is then recovered after a series of analytical procedures. Yet for water matrices such as wastewater, due to the unknown background concentrations of targeted viruses in the matrix and the variable recovery efficiency between individual processes, only an approximation of the recovery efficiency may be obtained from such spike-and-recovery experiments. In this study, we demonstrated theoretically that for two widely used approximations, the error in estimating virus recovery should be less than the ratio of the amount of target virus in the background sample to that in the spike. Furthermore, we developed an applicable method, based on this new understanding, for deciding on the amount of virus for spiking before conducting a spike-and-recovery experiment, so that the approximation error is restricted to an acceptable level for each individual process. Finally, we applied the method to a set of experimental data for viruses in wastewater, demonstrating its utility and noting its general applicability to other pathogens or water matrices. IMPORTANCE The performance of procedures for pathogen log reduction is at the heart of new risk-based guidance/regulation globally, yet the methods for undertaking assessments of pathogen recovery are not standardized despite their fundamental impacts on assessing log reductions. Here we describe the level of spiking agent(s) that is necessary to correctly assess spiked pathogen/surrogate recovery with whatever method is deployed. The significance of our research lies in identifying the importance of the amount of spiking agents for reducing uncertainty in recovery estimates, which will allow the development of a recommendation for spiking experiments, proactively applying this understanding.

Assessing human‐specific CrAssphage recovery after acidification‐filtration concentrating method in environmental water

“…Moreover, background level of crAssphage is one of the important factors that could affect the recovery estimation using the spiking method (Li et al, ). A high abundance of crAssphage was detected in the WWTP inf , WWTP eff , and canal samples (CR usp ).…”

“…The reduced levels of crAssphage could be due to loss during the filtration step, where no suspended solids or organic matters were present in PBS to enhance filtration recovery. Consequently, the difference between recoveries in PBS and recoveries in environmental samples underlines the importance of evaluating method recovery in real environmental samples that reflect actual matrix effect (Li et al, ).…”

“…Approximation of crAssphage recovery followed this formula: recovery ( R ) = (CR sp − CR usp )/CR st , where CR sp is gene copies of recovered crAssphage in a spiked reactor, CR usp is gene copies of recovered crAssphage in an unspiked reactor, and CR st is gene copies of crAssphage spiked into the reactors (Li, Qiu, Pang, & Ashbolt, ; US EPA, ). The calculating equation was used by assuming that equal recovery loss occurred in spiked and unspiked reactors.…”

“…The calculating equation was used by assuming that equal recovery loss occurred in spiked and unspiked reactors. Error of approximation was calculated as CR usp /(CR st × R PBS ), where R PBS is the crAssphage recovery in PBS (Li et al, ).…”

Capsid Integrity qPCR—An Azo-Dye Based and Culture-Independent Approach to Estimate Adenovirus Infectivity after Disinfection and in the Aquatic Environment

“…For researchers in lower resource settings without access to enteric virus cell-culture assays, there seems to be considerable value in utilizing ci-qPCR to estimate the concentration of potentially infectious enteric viruses but noting the inability to assay UV-treated samples. Given the generally high persistence of viral pathogens both in the aquatic [46,54] and wastewater environments [80][81][82][83], and that enteric virus infection risks to dominate in wastewater reuse applications [84], e.g., for irrigation purposes, the removal of "false-positive" infectious virus results has the potential to increase the significance of qPCR results for public health, economic, and QMRA purposes.…”