Feasibility of neighborhood and building scale wastewater-based genomic epidemiology for pathogen surveillance

Abstract

The benefits of wastewater-based epidemiology (WBE) for tracking the viral load of SARS-CoV-2, the causative agent of COVID-19, have become apparent since the start of the pandemic. However, most sampling occurs at the wastewater treatment plant influent and therefore monitors the entire catchment, encompassing multiple municipalities, and is conducted using quantitative polymerase chain reaction (qPCR), which only quantifies one target. Sequencing methods provide additional strain information and also can identify other pathogens, broadening the applicability of WBE to beyond the COVID-19 pandemic. Here we demonstrate feasibility of sampling at the neighborhood or building complex level using qPCR, targeted sequencing, and untargeted metatranscriptomics (total RNA sequencing) to provide a refined understanding of the local dynamics of SARS-CoV-2 strains and identify other pathogens circulating in the community. We demonstrate feasibility of tracking SARS-CoV-2 at the neighborhood, hospital, and nursing home level with the ability to detect one COVID-19 positive out of 60 nursing home residents. The viral load obtained was correlative with the number of COVID-19 patients being treated in the hospital. Targeted wastewater-based sequencing over time demonstrated that nonsynonymous mutations fluctuate in the viral population. Clades and shifts in mutation profiles within the community were monitored and could be used to determine if vaccine or diagnostics need to be adapted to ensure continued efficacy. Furthermore, untargeted RNA sequencing identified several other pathogens in the samples. Therefore, untargeted RNA sequencing could be used to identify new outbreaks or emerging pathogens beyond the COVID-19 pandemic.

Keywords: Mutation analysis; RNAseq; SARS-CoV-2; Targeted genome sequencing; Viral load; Wastewater based epidemiology.

Graphical abstract

Fig. 1

Processing flowchart for Total RNA-Seq data. RNA-Seq data was obtained from runs on an Illumina NextSeq instrument. Files were demultiplexed, quality checked, and trimmed. Sequences were assembled using SPAdes into contigs and then compared to known reference sequences in the refseq database using BlastN or DIAMOND BlastX to identify pathogens in the wastewater samples.

Fig. 2

Sampling sites within Toledo, Ohio, USA. Orange circles indicate manholes sampled. Brown lines indicate sewer lines. A. Hospital P. B. Hospital V. C. Community C. D. Nursing home N. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Relative abundance of viruses, bacteria, archaea, and eukaryotes identified by total RNA sequencing of wastewater. Collection dates are indicated as 1 (7/14/20), 2 (7/21/20), or 3 (7/28/20).

Fig. 4

Ecological niches associated with the microorganisms identified by total RNA sequencing identified several human, animal and pl ant pathogens, as well as potential and opportunistic pathogens. Sample sites are indicated as Nursing home N (N), Hospital V (V), Hospital P (P), and the Community C (C). Collection dates are indicated as 1 (7/14/20), 2 (7/21/20), or 3 (7/28/20).

Fig. 5

The abundance of pathogens identified by total RNA sequencing varied by sampling site and over time. Notably, pathogens associated with nosocomial infections were found in the effluent from the Nursing Home (N) and Hospital V (V), but not in Hospital P (P) or the Community C (C). Weeks of collection are indicated as 1 (7/14/20), 2 (7/21/20), or 3 (7/28/20).