A comparison of precipitation and fi ltration-based SARS-CoV-2 recovery methods and the in fl uence of temperature, turbidity, and surfactant load in urban wastewater of the Total Environment

has become a complimentary surveillance tool during the SARS-CoV-2 pan- demic. Viral concentration methods from wastewater are still being optimised and compared, whilst viral recovery under different wastewater characteristics and storage temperatures remains poorly understood. Using urban waste- water samples, we tested three viral concentration methods; polyethylene glycol precipitation (PEG), ammonium sul-phateprecipitation(AS), andCP select ™ InnovaPrep® (IP) ultra fi ltration. WefoundnomajordifferenceinSARS-CoV- 2 and faecal indicator virus (crAssphage) recovery from wastewater samples ( n = 46) using these methods, PEG slightly (albeit non-signi fi cantly), outperformed AS and IP for SARS-CoV-2 detection, as a higher genome copies per litre (gc/l) was recorded for a larger proportion of samples. Next generation sequencing of 8 paired samples revealed non-signi fi cant differencesin thequalityofdatabetweenASandIP, thoughIPdataqualitywasslightlybetterandless variable. A controlledexperiment assessed the impact ofwastewatersuspended solids (turbidity; 0 – 400 NTU), surfac-tantload(0 – 200mg/l),andstoragetemperature(5 – 20°C)onviralrecoveryusingtheASandIPmethods.SARS-CoV-2 recoverieswere > 20%withASand < 10%withIPinturbidsamples,whilstviralrecoveriesforsampleswithadditional surfactant were between 0 – 18% for AS and 0 – 5% for IP. Turbidity and sample storage temperature combined had no signi fi canteffectonSARS-CoV-2recovery( p > 0.05),whilstsurfactantandstoragetemperaturecombinedweresignif-icant negativecorrelates( p < 0.001and p < 0.05, respectively).Inconclusion, our results showthat choiceofmethod- ology had small effect on viral recovery of SARS-CoV-2 and crAssphage in wastewater samples within this study. In contrast, sample turbidity, storage temperature, and surfactant load did affect viral recovery, highlighting the need for careful consideration of the viral concentration methodology used when working with wastewater samples.

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