Conference Agenda

Wastewater-based epidemiology (WBE) has been utilized to track community infections of COVID-19 by detecting RNA of the virus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), within samples collected from wastewater. As SARS-CoV-2 has been evolving through the emergence of new variants, the relationships between community infections and wastewater measurements of the RNA can potentially change. This study analyzed SARS-CoV-2 RNA, and indicators of human waste in wastewater from two sewersheds of different scales (University of Miami (UM) campus and Miami-Dade County Central District wastewater treatment plant (MDC-CDWWTP)) during five internally defined COVID-19 variant dominant periods (Initial wave, Pre-Delta wave, Delta wave, Omicron wave and Post-Omicron wave). SARS-CoV-2 RNA quantities were compared against COVID-19 clinical cases and hospitalizations to evaluate correlations with wastewater SARS-CoV-2 RNA levels during the different variant periods. Results show that although the correlations between documented clinical cases and hospitalizations were high, the prevalence for a given wastewater SARS-CoV-2 level varied depending upon the variant analyzed. The correlative relationship between the levels in wastewater and clinical cases was significantly steeper (more cases per level found in wastewater) for the omicron-dominated period relative to other periods, while for non-omicron-dominated periods the relationship between wastewater level and clinical cases was flatter. For hospitalization, the relationships were steepest for the initial wave, followed by the delta wave with flatter slopes during all other waves. Overall results were interpreted in the context of SARS-CoV-2 virulence and vaccination rates among the community.

Antibiotic resistance (AR) poses a severe threat to public health. With the massive use of a last-resort antibiotics, carbapenems, clinical reports of the infections associated with carbapenem-resistant Enterobacterales (CRE) have increased. Clinical surveillance involves susceptibility testing and/or whole genome sequencing of resistant isolates, which is laborious and requires expertise and resources. Wastewater surveillance can potentially complement clinical surveillance of AR. To characterize AR in wastewater, culturing antibiotic resistant bacteria and quantification of antibiotic resistance genes (ARGs) are the two most widely used methods. However, few studies have compared the two methods, limiting our ability to compare AR surveillance results across studies. To address this knowledge gap, we assessed the associations between the quantitative results generated by (1) a culture-based quantification of CRE, and (2) droplet digital PCR quantification of five representative ARGs encoding for carbapenemase-production (CP). To elaborate, total CRE, and its constituent population, carbapenemase-producing Enterobacterales (CP-CRE), were quantified in wastewater samples over twelve weeks and three sites using a stepwise culture-based method. In parallel, a multiplexed ddPCR assay targeting five CP genes (blaKPC, blaVIM, blaIMP, blaNDM and blaOXA-48-like) was performed on the same wastewater samples. Weak correlations were observed between CFU of total CRE, CP-CRE and ARG abundance (copies/mL-wastewater). This result indicated that discretion should be used when comparing the “signal” of AR across wastewater samples when using different methods. Current efforts involve characterizing AR with sequencing to investigate the observed discrepancy between culture- and ddPCR-based results. This study highlights the importance of method standardization of AR wastewater surveillance.

The COVID-19 pandemic has highlighted the value of environmental surveillance of viruses for public health. Wastewater-based epidemiology (WBE) is now widely used to monitor SARS-CoV-2, influenza, RSV, norovirus, Mpox, and other viruses in communities. To date, there has been little research on virus genome stability in wastewater, especially on how genome type and encapsidation may affect genome stability. In this study, we used four model viruses to investigate the effect of the capsid on genome signal and compare viral RNA and DNA persistence in untreated wastewater. We found that encapsidated viral DNA is more persistent than RNA in wastewater and that virus infectivity inactivates more quickly than genome signal. This research demonstrates that the virus capsid provides significant protection to the nucleic acid signals that we commonly rely on for WBE. Further, we demonstrate the fast degradation kinetics of extraviral RNA and DNA when the capsid is not present. Our data also probes the effect of different amplicon sizes used to measure nucleic acid signals and show that the amplicon sizes employed in measurements are not having a major effect on the quantities of gene copies measured over the range we tested (99-395 bp). Although wastewater-based surveillance has been successful for SARS-CoV-2 during the global pandemic, this study on both RNA and DNA builds an important knowledge base for all viruses with different genome types and structures. These findings will be valuable for interpreting viral nucleic acid measurements in wastewater and specifically how they inform public health and water safety.

Despite the advantages of wastewater based surveillance (WBS) for monitoring community health, it has been used primarily in densely populated communities with relatively well-maintained and funded sewer infrastructure. Consequently, there is a lack of knowledge base on how rural communities in the U.S. can take advantage of WBS to understand existing health disparities and subsequently design more targeted public health interventions. In this study, we initiated a year-long wastewater monitoring campaign in a small, rural sewershed in Southwest Virginia with known infrastructural challenges, including sewer main leaks and groundwater and surface water inflow and infiltration (I&I). In addition to collecting wastewater treatment plant (WWTP) influent, we sampled 12 sewershed nodes at each branch line to the sewer conveyance system to assess pathogen signal, and potential signal loss due to I&I and sewer system design characteristics. TaqMan Array Card (TAC) qPCR was performed for simultaneous quantification of 54 enteric pathogen targets. After six months of data collection, Giardia lamblia, Norovirus, and Adenovirus were present in 86%, 11%, and 32% of WWTP influent samples respectively. Due to a 22% lower detection rate of pathogen signal at the influent as compared to downstream of a large residential facility, preliminary results point to the risk of underestimating pathogen circulation when sampling only WWTP influent in a system with persistent I&I, informing design considerations for WBS implementation in rural areas.

Cryptosporidium, a parasitic protozoan, is a major enteric pathogen of public health concern and globally recognized as a significant cause of waterborne diseases. Approximately one million people in the Portland metro area receive unfiltered drinking water from the Bull Run Watershed and are at increased risk for cryptosporidiosis outbreaks. Passive case surveillance for cryptosporidiosis is estimated to identify <2% of cases due to asymptomatic and mildly symptomatic infections as well as under-reporting by clinicians. As infected individuals shed 10⁷-10⁹ Cryptosporidium oocysts per bowel movement, wastewater surveillance of Cryptosporidium may be an effective public health tool to identify cryptosporidiosis risk and notify public works of drinking water contamination. There currently are no standardized wastewater concentration methods or molecular techniques to detect and quantify Cryptosporidium in wastewater. This results in highly variable detection limits and specificity, making it difficult to compare results between studies. This study utilized wastewater spiked with Cryptosporidium oocysts to evaluate eight DNA extraction protocols utilizing two commercial kits with and without bead beating and freeze-thawing cycles, and 12 oocyst concentration protocols including centrifugation, electronegative membrane filtration, and Envirocheck filtration, at various wastewater volumes. The recovery efficiency of each protocol was assessed through qPCR using a TaqMan assay targeting the Cryptosporidium 18S rRNA gene. PCR products were sequenced to assess the specificity of each concentration protocol used. The validated methods were utilized to quantify wastewater Cryptosporidium concentrations in three diverse Oregon communities (Corvallis, Bend and Portland) over time.