Human pathogen nucleic acids in wastewater solids from 191 wastewater treatment plants in the United States

Abstract

We measured concentrations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, influenza A and B viruses, respiratory syncytial virus, human metapneumovirus, enterovirus D68, human parainfluenza types 1, 2, 3, 4a, and 4b in aggregate, norovirus genotype II, rotavirus, Candida auris, hepatitis A virus, human adenovirus, mpox virus, H5 influenza A virus, and pepper mild mottle virus nucleic acids in wastewater solids prospectively at 191 wastewater treatment plants in 40 states across the United States plus Washington DC. Measurements were made two to seven times per week from 1 January 2022 to 30 June 2024, depending on wastewater treatment plant staff availability. Measurements were made using droplet digital (reverse-transcription-) polymerase chain reaction (ddRT-PCR) following best practices for making environmental molecular biology measurements. These data can be used to better understand disease occurrence in communities contributing to the wastewater.

Fig. 1

Map of wastewater treatment plants (WWTPs) enrolled in this study. States where there are enrolled sites are shaded in light purple. Each dot (191) represents a participating WastewaterSCAN site.

Fig. 2

Timeline of when different targets were measured during the study.

Fig. 3

Wastewater treatment plant enrollment count over the study period.

Fig. 4

Distribution of populations served by the 191 wastewater treatment plants who participated in the study. The y-axis represents the binned population served and the x-axis represents the number of WWTPs in each bin. “k” after the number indicates times 10³ and “M” after the number indicates times 10⁶.

Fig. 5

Arrangement of multiplexed PCR plates for pathogen targets run in the study. There are two distinct plate arrangements (A and B). Arrangement A was used for the majority of sites and arrangement B was used for a small subset of, at most, 16 WWTPs (see Table S1 when and where 10 replicates were run) followed their own arrangement. Each box represents a schematic of how assays were multiplexed during different periods of the project. The date at the bottom left side of each box is the start date and the date near the right edge of the box is the approximate end date (based on the date the assay was stopped in the lab, the date associated with the last sample run could be different depending on the date it was processed in the lab). All probes contained fluorescent molecules (FM) and quenchers (5′ FM/ZEN/3′ IBFQ), as indicated. FAM, 6-fluorescein amidite; HEX, hexachloro-fluorescein; ATTO590; ROX, carboxyrhodamine; Cy5, Cyanine5; Cy5.5, Cyanine5; and ZEN, a proprietary internal quencher from IDT (Coralville, Iowa); IBFQ, Iowa Black FQ. If the box is light gray then the annealing temperature is 59 °C and if it is dark gray, it is 61 °C. Assay abbreviations are provided in Tables 1 and 2 except for the following: N and S are assays targeting those genes in SARS-CoV-2. HV69-70 is the assay targeting the deletion 69/70 in the S gene characteristic BA.4 + BA.5 + BQ*. del143/145 is the assay targeting the 143/145 deletion in the S gene characteristics of SARS-CoV-2 BA.1. del156/157 is the assay targeting the deletion 156/157 in the S gene characteristic of SARS-CoV-2 Delta. BA.4 is the assay targeting the deletion 141/143 in the ORF1a gene characteristic of SARS-CoV-2 BA.4. BA.2 is the assay targeting the set of deletions LPPA24S characteristic of BA.2 + BA.4 + BA.5. XBB is the assay targeting adjacent SNPs in the S gene characteristic of SARS-CoV-2 XBB. XBB* is the assay targeting adjacent SNPs in the S gene characteristic of SARS-CoV-2 XBB*. HPIV are the assays targeting human parainfluenza 1, 2, 3, 4a, and 4b. The dashed yellow line indicates when instrumentation switched from QX200 to QX600.

Fig. 6

Example inhibition titrations for nine assays used for the study. The concentration of solids from wastewater solids samples placed in DNA/RNA Shield in the preanalytical step prior to homogenization and RNA extraction is shown on the x-axis. On the y-axis is the concentration of the target measured in the solids in units of copies per gram (cp/g) dry weight, corrected for the concentration of solids placed in the DNA/RNA shield. If a symbol is on the value of “0”, it means the result was “not detected”. This occurred in some cases when the mass of solids added to the DNA/RNA shield was small, thereby reducing the sensitivity of the method. Errors are shown as standard deviations across 6 replicates as described in the methods section. If error bars are not visible it is because they are smaller than the symbol.