Human viral nucleic acids concentrations in wastewater solids from Central and Coastal California USA

Abstract

We measured concentrations of SARS-CoV-2, influenza A and B virus, respiratory syncytial virus (RSV), mpox virus, human metapneumovirus, norovirus GII, and pepper mild mottle virus nucleic acids in wastewater solids at twelve wastewater treatment plants in Central California, USA. Measurements were made daily for up to two years, depending on the wastewater treatment plant. Measurements were made using digital droplet (reverse-transcription-) polymerase chain reaction (RT-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

The time period during which each of the 12 plants were enrolled in the study. Each plant is a row on the y-axis, and date is on the x-axis. When a sample was collected, gray is shown; white indicates no sample collected. The abbreviation for each plant is provided in Table 1.

Fig. 2

Maps of sewersheds of wastewater treatment plants enrolled in this study.

Fig. 3

Arrangement of multiplexed PCR plates for human viral targets run in the study. 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 and quenchers (5′ FAM and/or HEX/ZEN/3′ IBFQ), as indicated. FAM, 6-fluorescein amidite; HEX, hexachloro-fluorescein; ZEN, a proprietary internal quencher from IDT; IBFQ, Iowa Black FQ. If box is white, annealing temperature is 59 °C and if gray, it is 61 °C. N, S, and ORF1a are assays targeting those genes in SARS-CoV-2. del156/157 is the assay targeting the 156/157 deletion in the S gene characteristic of the Delta variant of SARS-CoV-2. HV69-70 is the assay targeting the deletion 69/70 in the S gene characteristic of Alpha, and various Omicron variants. del143/145 is the assay targeting the 143/145 deletion in the S gene characteristics of BA.1 Omicron. BA.4 is the assay targeting the deletion 141/143 in the ORF1a gene characteristic of BA.4. BA.2.75 is the assay targeting the adjunct SNPS (S:147E and) in the S gene characteristic of BA.2.75. IAV is the assay targeting a gene in the influenza A genome. RSV is the assay targeting a gene in the RSV genome. BA.2 is the assay targeting the set of deletions LPPA24S characteristic of BA.2. IBV is the assay targeting influenza B. MPXV is the assay targeting genes in MPOX virus. HMPV is the assay targeting genes in human metapneumovirus.

Fig. 4

Bovine coronavirus recovery at each plant. The lower and upper edges of the boxes are the 25th and 75th percentiles, respectively. The middle line in each box is the median. The lower and upper “whiskers” end at the 9th and 91st percentiles. The number of recoveries used for each plant are provided in Table 1. Abbreviations for each plant are shown on the x-axis. Multiple the factional values by 100 to obtain recovery as a percent.

Fig. 5

Example inhibition titrations for eight 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 68% confidence intervals across 10 replicates as described in the methods section. If error bars are not visible it is because they are smaller than the symbol.