Stanford Advisors

All Publications

  • Tools for interpretation of wastewater SARS-CoV-2 temporal and spatial trends demonstrated with data collected in the San Francisco Bay Area. Water research X Greenwald, H. D., Kennedy, L. C., Hinkle, A., Whitney, O. N., Fan, V. B., Crits-Christoph, A., Harris-Lovett, S., Flamholz, A. I., Al-Shayeb, B., Liao, L. D., Beyers, M., Brown, D., Chakrabarti, A. R., Dow, J., Frost, D., Koekemoer, M., Lynch, C., Sarkar, P., White, E., Kantor, R., Nelson, K. L. 2021; 12: 100111


    Wastewater surveillance for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA can be integrated with COVID-19 case data to inform timely pandemic response. However, more research is needed to apply and develop systematic methods to interpret the true SARS-CoV-2 signal from noise introduced in wastewater samples (e.g., from sewer conditions, sampling and extraction methods, etc.). In this study, raw wastewater was collected weekly from five sewersheds and one residential facility. The concentrations of SARS-CoV-2 in wastewater samples were compared to geocoded COVID-19 clinical testing data. SARS-CoV-2 was reliably detected (95% positivity) in frozen wastewater samples when reported daily new COVID-19 cases were 2.4 or more per 100,000 people. To adjust for variation in sample fecal content, four normalization biomarkers were evaluated: crAssphage, pepper mild mottle virus, Bacteroides ribosomal RNA (rRNA), and human 18S rRNA. Of these, crAssphage displayed the least spatial and temporal variability. Both unnormalized SARS-CoV-2 RNA signal and signal normalized to crAssphage had positive and significant correlation with clinical testing data (Kendall's Tau-b (tau)=0.43 and 0.38, respectively), but no normalization biomarker strengthened the correlation with clinical testing data. Locational dependencies and the date associated with testing data impacted the lead time of wastewater for clinical trends, and no lead time was observed when the sample collection date (versus the result date) was used for both wastewater and clinical testing data. This study supports that trends in wastewater surveillance data reflect trends in COVID-19 disease occurrence and presents tools that could be applied to make wastewater signal more interpretable and comparable across studies.

    View details for DOI 10.1016/j.wroa.2021.100111

    View details for PubMedID 34373850

  • Sewage, Salt, Silica, and SARS-CoV-2 (4S): An Economical Kit-Free Method for Direct Capture of SARS-CoV-2 RNA from Wastewater ENVIRONMENTAL SCIENCE & TECHNOLOGY Whitney, O. N., Kennedy, L. C., Fan, V., Hinkle, A., Kantor, R., Greenwald, H., Crits-Christoph, A., Al-Shayeb, B., Chaplin, M., Maurer, A. C., Tjian, R., Nelson, K. L. 2021; 55 (8): 4880-4888


    Wastewater-based epidemiology is an emerging tool to monitor COVID-19 infection levels by measuring the concentration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater. There remains a need to improve wastewater RNA extraction methods' sensitivity, speed, and reduce reliance on often expensive commercial reagents to make wastewater-based epidemiology more accessible. We present a kit-free wastewater RNA extraction method, titled "Sewage, Salt, Silica and SARS-CoV-2" (4S), that employs the abundant and affordable reagents sodium chloride (NaCl), ethanol, and silica RNA capture matrices to recover sixfold more SARS-CoV-2 RNA from wastewater than an existing ultrafiltration-based method. The 4S method concurrently recovered pepper mild mottle virus (PMMoV) and human 18S ribosomal subunit rRNA, which have been proposed as fecal concentration controls. The SARS-CoV-2 RNA concentrations measured in three sewersheds corresponded to the relative prevalence of COVID-19 infection determined via clinical testing. Lastly, controlled experiments indicate that the 4S method prevented RNA degradation during storage of wastewater samples, was compatible with heat pasteurization, and in our experience, 20 samples can be processed by one lab technician in approximately 2 h. Overall, the 4S method is promising for effective, economical, and accessible wastewater-based epidemiology for SARS-CoV-2, providing another tool to fight the global pandemic.

    View details for DOI 10.1021/acs.est.0c08129

    View details for Web of Science ID 000643546400068

    View details for PubMedID 33759506

    View details for PubMedCentralID PMC8009096

  • Challenges in Measuring the Recovery of SARS-CoV-2 from Wastewater ENVIRONMENTAL SCIENCE & TECHNOLOGY Kantor, R. S., Nelson, K. L., Greenwald, H. D., Kennedy, L. C. 2021; 55 (6): 3514-3519


    Wastewater-based epidemiology is an emerging tool for tracking the spread of SARS-CoV-2 through populations. However, many factors influence recovery and quantification of SARS-CoV-2 from wastewater, complicating data interpretation. Specifically, these factors may differentially affect the measured virus concentration, depending on the laboratory methods used to perform the test. Many laboratories add a proxy virus to wastewater samples to determine losses associated with concentration and extraction of viral RNA. While measuring recovery of a proxy virus is an important process control, in this piece, we describe the caveats and limitations to the interpretation of this control, including that it typically does not account for losses during RNA extraction. We recommend reporting the directly measured concentration data alongside the measured recovery efficiency, rather than attempting to correct the concentration for recovery efficiency. Even though the ability to directly compare SARS-CoV-2 concentrations from different sampling locations determined using different methods is limited, concentration data (uncorrected for recovery) can be useful for public health response.

    View details for DOI 10.1021/acs.est.0c08210

    View details for Web of Science ID 000636727200008

    View details for PubMedID 33656856

  • Genome Sequencing of Sewage Detects Regionally Prevalent SARS-CoV-2 Variants. mBio Crits-Christoph, A. n., Kantor, R. S., Olm, M. R., Whitney, O. N., Al-Shayeb, B. n., Lou, Y. C., Flamholz, A. n., Kennedy, L. C., Greenwald, H. n., Hinkle, A. n., Hetzel, J. n., Spitzer, S. n., Koble, J. n., Tan, A. n., Hyde, F. n., Schroth, G. n., Kuersten, S. n., Banfield, J. F., Nelson, K. L. 2021; 12 (1)


    Viral genome sequencing has guided our understanding of the spread and extent of genetic diversity of SARS-CoV-2 during the COVID-19 pandemic. SARS-CoV-2 viral genomes are usually sequenced from nasopharyngeal swabs of individual patients to track viral spread. Recently, RT-qPCR of municipal wastewater has been used to quantify the abundance of SARS-CoV-2 in several regions globally. However, metatranscriptomic sequencing of wastewater can be used to profile the viral genetic diversity across infected communities. Here, we sequenced RNA directly from sewage collected by municipal utility districts in the San Francisco Bay Area to generate complete and nearly complete SARS-CoV-2 genomes. The major consensus SARS-CoV-2 genotypes detected in the sewage were identical to clinical genomes from the region. Using a pipeline for single nucleotide variant calling in a metagenomic context, we characterized minor SARS-CoV-2 alleles in the wastewater and detected viral genotypes which were also found within clinical genomes throughout California. Observed wastewater variants were more similar to local California patient-derived genotypes than they were to those from other regions within the United States or globally. Additional variants detected in wastewater have only been identified in genomes from patients sampled outside California, indicating that wastewater sequencing can provide evidence for recent introductions of viral lineages before they are detected by local clinical sequencing. These results demonstrate that epidemiological surveillance through wastewater sequencing can aid in tracking exact viral strains in an epidemic context.

    View details for DOI 10.1128/mBio.02703-20

    View details for PubMedID 33468686

  • Effect of disinfectant residual, pH, and temperature on microbial abundance in disinfected drinking water distribution systems ENVIRONMENTAL SCIENCE-WATER RESEARCH & TECHNOLOGY Kennedy, L. C., Miller, S. E., Kantor, R. S., Nelson, K. L. 2021; 7 (1): 78-92

    View details for DOI 10.1039/d0ew00809e

    View details for Web of Science ID 000605449700006

  • CNC-loaded hydrogel particles generated from single-and double-emulsion drops GREEN MATERIALS Ye, C., Kennedy, L., Shirk, K., Cordova-Figueroa, U. M., Youngblood, J., Martinez, C. J. 2015; 3 (1): 25-34