pubmed.ncbi.nlm.nih.gov

Comparative Assessment of Filtration- and Precipitation-Based Methods for the Concentration of SARS-CoV-2 and Other Viruses from Wastewater - PubMed

  • ️Sat Jan 01 2022

Comparative Assessment of Filtration- and Precipitation-Based Methods for the Concentration of SARS-CoV-2 and Other Viruses from Wastewater

Kata Farkas et al. Microbiol Spectr. 2022.

Abstract

Wastewater-based epidemiology (WBE) has been widely used to track levels of SARS-CoV-2 infection in the community during the COVID-19 pandemic. Due to the rapid expansion of WBE, many methods have been used and developed for virus concentration and detection in wastewater. However, very little information is available on the relative performance of these approaches. In this study, we compared the performance of five commonly used wastewater concentration methods for the detection and quantification of pathogenic viruses (SARS-CoV-2, norovirus, rotavirus, influenza, and measles viruses), fecal indicator viruses (crAssphage, adenovirus, pepper mild mottle virus), and process control viruses (murine norovirus and bacteriophage Phi6) in laboratory spiking experiments. The methods evaluated included those based on either ultrafiltration (Amicon centrifugation units and InnovaPrep device) or precipitation (using polyethylene glycol [PEG], beef extract-enhanced PEG, and ammonium sulfate). The two best methods were further tested on 115 unspiked wastewater samples. We found that the volume and composition of the wastewater and the characteristics of the target viruses greatly affected virus recovery, regardless of the method used for concentration. All tested methods are suitable for routine virus concentration; however, the Amicon ultrafiltration method and the beef extract-enhanced PEG precipitation methods yielded the best recoveries. We recommend the use of ultrafiltration-based concentration for low sample volumes with high virus titers and ammonium levels and the use of precipitation-based concentration for rare pathogen detection in high-volume samples. IMPORTANCE As wastewater-based epidemiology is utilized for the surveillance of COVID-19 at the community level in many countries, it is crucial to develop and validate reliable methods for virus detection in sewage. The most important step in viral detection is the efficient concentration of the virus particles and/or their genome for subsequent analysis. In this study, we compared five different methods for the detection and quantification of different viruses in wastewater. We found that dead-end ultrafiltration and beef extract-enhanced polyethylene glycol precipitation were the most reliable approaches. We also discovered that sample volume and physico-chemical properties have a great effect on virus recovery. Hence, wastewater process methods and start volumes should be carefully selected in ongoing and future wastewater-based national surveillance programs for COVID-19 and beyond.

Keywords: enteric viruses; environmental virology; human respiratory viruses; public health surveillance; sewage concentration.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1

Greater recovery of spiked viruses, influenza A/B viruses (flu-A/B), measles virus (MeV), murine norovirus (MNV), SARS-CoV-2 (N1), norovirus GII (NoVGII), bacteriophage phi6 (Phi6), and rotavirus (RoV) in deionized water (DW) compared to wastewater (WW). Data derived from all concentration methods. (a) All spiked virus recovery results combined. (b) Recovery by individual virus. Boxes depict the 25th, 50th, and 75th percentile ranges after omitting outliers greater or less than ±1.5× the interquartile range (IQR), which is shown by the whiskers.

FIG 2
FIG 2

Recovery for human mastadenovirus (AdV), crAssphage (CrAss), influenza A/B virus (Flu-A/B), measles virus (MeV), murine norovirus (MNV), SARS-CoV-2 (N1), norovirus GII (NoVGII), bacteriophage phi6 (Phi6), pepper mild mottle virus (PMMoV), and rotavirus (RoV) as a function of starting volume of wastewater. Data derived from all concentration methods. (a) All recovery results combined. (b) Recovery separated by virus with a variable y scale (recovery percentage). (c) Existence of any significant differences in the tested volumes. (To analyze which volume is significantly better for viral recovery, panel c is to be analyzed in conjunction with panel ‘a and panel b). The P values (Holm adjustment method) of pairwise comparisons were calculated between extraction volumes with two sample t tests with pooled standard deviations (***, P < 0.001; **, P < 0.01; *, P < 0.05). Comparisons were made with an ANOVA after log10 transformation of recovery, followed by pairwise t tests; Pairwise comparisons found significant differences between all volumes with the Holm adjustment method (P < 0.05).

FIG 3
FIG 3

Influence of concentration methods on virus recovery from wastewater at a sample starting volume of 15 mL. (a) All recovery results with starting volumes of 15 mL combined. (b) Recovery separated by virus with a variable y scale. (c) Existence of any significant differences among concentration methods. (To analyze which volume is significantly better for viral recovery, panel c is to be analyzed in conjunction with panel a and panel b). The P values of pairwise comparisons of method recovery were calculated using t tests without pooled standard deviations (***, P < 0.001; **, P < 0.01; *, P < 0.05; -., P > 0.05; P value Holm adjustment method). Comparisons were made with an ANOVA after log10 transformation of recovery, followed by pairwise t tests (c); BE-PEG and Amicon methods had the highest median recovery, but due to Amicon method’s greater variance, pairwise comparisons with IP (third-highest median recovery) were only significantly different for BE-PEG (P < 0.05; panel c).

FIG 4
FIG 4

Viral recovery in the pellet from the first centrifugation step (10,000 × g, 10 min, 4°C) in the viral extraction procedure. The sample solid fraction (pellet) has significantly lower viral recovery than the concentrated sample; thus, removal via centrifugation will likely increase the median viral recovery of a concentrated sample. (a) All recovery results with starting volumes of 50 mL combined. (b) Recovery separated by virus with a variable y scale. Comparisons were made with a Welch two-sample t test after log10 transformation of recovery.

FIG 5
FIG 5

Comparison of viral recovery for Amicon and BE-PEG concentration methods tested on neat, unspiked wastewater samples collected at 13 wastewater treatment plants. Statistical comparisons were made using paired t tests after log transformation of the gene copies per liter. Recovery of crAss, enterovirus (EV), and enterovirus D68 (EVD68) could not be assumed to have differing means, while SARS-CoV-2 and norovirus GI (NoVGI) had significantly greater mean recovery with the Amicon method, and NoVGII had significantly greater mean recovery with the BE-PEG method.

Similar articles

Cited by

References

    1. WHO. 2020. WHO Coronavirus (COVID-19) dashboard. https://covid19.who.int/. Accessed 15 February 2022.
    1. Bhat EA, Khan J, Sajjad N, Ali A, Aldakeel FM, Mateen A, Alqahtani MS, Syed R. 2021. SARS-CoV-2: insight in genome structure, pathogenesis and viral receptor binding analysis: an updated review. Int Immunopharmacol 95:107493. doi:10.1016/j.intimp.2021.107493. - DOI - PMC - PubMed
    1. Pascarella G, Strumia A, Piliego C, Bruno F, del Buono R, Costa F, Scarlata S, Agrò FE. 2020. COVID‐19 diagnosis and management: a comprehensive review. J Intern Med 288:192–206. doi:10.1111/joim.13091. - DOI - PMC - PubMed
    1. Oran DP, Topol EJ. 2021. The proportion of SARS-CoV-2 infections that are asymptomatic: a systematic review. Ann Intern Med 174:655–662. doi:10.7326/M20-6976. - DOI - PMC - PubMed
    1. Mahajan A, Solanki R, Sivadas N. 2021. Estimation of undetected symptomatic and asymptomatic cases of COVID‐19 infection and prediction of its spread in the USA. J Med Virol 93:3202–3210. doi:10.1002/jmv.26897. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances