Impact of sample clarification by size exclusion on virus detection and diversity in wastewater-based epidemiology.

The use of wastewater-based epidemiology (WBE) for early detection of virus circulation and response during the SARS-CoV-2 pandemic increased interest in and use of virus concentration protocols that are quick, scalable, and efficient. One such protocol involves sample clarification by size fractionation using either low-speed centrifugation to produce a clarified supernatant or membrane filtration to produce an initial filtrate depleted of solids, eukaryotes and bacterial present in wastewater (WW), followed by concentration of virus particles by ultrafiltration of the above. While this approach has been successful in identifying viruses from WW, it assumes that majority of the viruses of interest should be present in the fraction obtained by ultrafiltration of the initial filtrate, with negligible loss of viral particles and viral diversity. We used WW samples collected in a population of ~700,000 in southwest USA between October 2019 and March 2021, targeting three non-enveloped viruses (enteroviruses [EV], canine picornaviruses [CanPV], and human adenovirus 41 [Ad41]), to evaluate whether size fractionation of WW prior to ultrafiltration leads to appreciable differences in the virus presence and diversity determined. We showed that virus presence or absence in WW samples in both portions (filter trapped solids [FTS] and filtrate) are not consistent with each other. We also found that in cases where virus was detected in both fractions, virus diversity (or types) captured either in FTS or filtrate were not consistent with each other. Hence, preferring one fraction of WW over the other can undermine the capacity of WBE to function as an early warning system and negatively impact the accurate representation of virus presence and diversity in a population.

Genome Sequence of a Microvirus Recovered from Wastewater in Arizona, USA, in October 2020, Encodes a Previously Undescribed DNA-Binding Protein.

We describe the genome of Microvirus-AZ-2020, which was identified from wastewater in Arizona, USA, in October 2020. Microvirus-AZ-2020 belongs to subfamily Gokushovirinae and contains six (five known and one hypothetical) open reading frames (ORFs), each with >40 codons. HHPred analysis and Colabfold structure prediction suggest that the hypothetical ORF encodes a previously undescribed putative DNA-binding protein.

Mass trends of parabens, triclocarban and triclosan in Arizona wastewater collected after the 2017 FDA ban on antimicrobials and during the COVID-19 pandemic.

Antimicrobials like parabens, triclosan (TCS), and triclocarban (TCC) are of public health concern worldwide due to their endocrine-disrupting properties and ability to promote antimicrobial drug resistance in human pathogens. The overall use of antimicrobials presumably has increased during the COVID-19 pandemic, whereas TCS and TCC may have experienced reductions in use due to their recent ban from thousands of over-the-counter (OTC) personal care products by the U.S. Food and Drug Administration (FDA). No quantitative data are available on the use of parabens or the impact the FDA ban had on TCC and TCS. Here, we use wastewater samples (n = 1514) from 10 different communities in Arizona to measure the presence of the six different antimicrobial products (TCS, TCC, and four alkylated parabens [methylparaben (MePb), ethylparaben (EtPb), propylparaben (PrPb), butylparaben (BuPb)]) collected before and during the COVID-19 pandemic using a combination of solid-phase extraction, liquid chromatography/tandem mass spectrometry (LC-MS/MS), and isotope dilution for absolute quantitation. The average mass loadings of all antimicrobials combined (1,431 ± 22 mg/day per 1,000 people) after the onset of the local epidemic (March 2020 - October 2020) were significantly higher (945 ± 62 mg/day per 1,000 people; p < 0.05) than before the pandemic (January 2019 - February 2020). Overall, parabens (∑Pbs = 999 ± 16 mg/day per 1,000 people) were the most used antimicrobials, followed by TCS (117 ± 14 mg/day per 1,000 people) and TCC (117 ± 14 mg/day per 1,000 people). After the 2017 U.S. FDA ban, we found a statistically significant (p < 0.05) reduction in the mass loadings of TCS (-89%) and TCC (-80%) but a rise in paraben use (+72%). Mass flows of 3 of a total of 4 parabens (MePb, EtPb, and PrPb) in wastewater were significantly higher upon the onset of the epidemic locally (p < 0.05). This is the first longitudinal study investigating the use of antimicrobials during the COVID-19 pandemic by employing wastewater-based epidemiology. Whereas an overall increase in the use of antimicrobials was evident from analyzing Arizona wastewater, a notable reduction in the use of TCS and TCC was evident during the pandemic, triggered by the U.S. FDA ban.

Canine picornaviruses detected in wastewater in Arizona, USA 2019 and 2021.

Virus surveillance by wastewater-based epidemiology (WBE) in two Arizona municipalities in Maricopa County, USA (~700,000 people), revealed the presence of six canine picornavirus (CanPV) variants: five in 2019 and one in 2021. Phylogenetic analysis suggests these viruses might be from domestic dog breeds living within or around the area. Phylogenetic and pairwise identity analyses suggest over 15 years of likely enzootic circulation of multiple lineages of CanPV in the USA and possibly globally. Considering <10 CanPV sequences are publicly available in GenBank as of June 2, 2022, the results provided here constitute an increase of current knowledge on CanPV diversity and highlight the need for increased surveillance.