Covid 19: Pilot: WACKY: Wastewater Assessment for Coronavirus in Kentucky

Keck, James W WACKY: Wastewater Assessment for Coronavirus in Kentucky Significance: Surveillance for SARS-CoV2 is hampered by limited clinical testing in the US and internationally. Lack of SARS-CoV2 surveillance is detrimental to public health and health system preparedness and response activities. SARS-CoV2 is shed in the stool of infected people1 and has been detected in the wastewater of several urban areas.2–4 Presence of SARS-CoV2 RNA in wastewater correlated with clinically observed COVID-19 disease in Paris and Brisbane.2,3 Wastewater-based epidemiology (WBE) provides an alternative strategy for SARS-CoV2 surveillance by evaluating samples of wastewater for the presence of viral biomarkers.5,6 This approach overcomes several limitations of clinical surveillance, such as the need for robust healthcare and laboratory infrastructure and the lack of representative and comprehensive testing within communities. WBE methods typically use samples from sewer systems or wastewater treatment facilities that undergo RNA concentration, extraction, and cleansing steps prior to the use of advanced PCR technology to quantitate viral RNA. The current approach is time and resource-intensive which limits the wide-scale application of WBE. Further, viral RNA is prone to degradation, and current requirements of shipping to a central lab are limiting. Developing technology to simplify wastewater RNA collection will support the deployment of WBE for SARS- CoV2 in low- and high-resource settings. In lower-resource and rural settings human waste is not collected and treated by centralized facilities. Human waste from latrines, open defecation and porous septic and sewer systems contaminates environmental open water sources like streams and standing water. Environmental contamination of surface water with enteric viruses is widespread.7–9 Environmental testing of open water, particularly in low-income international communities could identify SARS-CoV2 in the community. In high- resource settings improved WBE technology could support the surveillance of high-risk congregate living settings like long-term care facilities or university dormitories by monitoring effluent from the building for SARS-CoV2. We propose developing technology to simplify the extraction and concentration of SARS-CoV2 RNA from wastewater and environmental surface water. As a secondary objective, we will measure the environmental stability of SARS-CoV2 RNA in water under various environmental conditions to inform the sensitivity of WBE for SARS-CoV2 surveillance. Our team is uniquely positioned to carry out this pilot project with proven expertise in developing user-friendly RNA-based diagnostic technology (Dr. Berry), community-engaged environmental and wastewater testing (Dr. Hobbs), and creating new infectious disease surveillance systems (Dr. Keck). We will employ new technology developed by Dr. Berry termed Exclusion-based Sample Preparation (ESP), which provides a fast, simple, and electricity-free means of manipulating RNA. As preliminary data, ESP was used in Uganda to extract HIV viral RNA from clinical samples, yielding quantitative viral load measurements that were identical (on metrics of accuracy, analytical sensitivity, and specificity) to those obtained with the sophisticated Abbott m2000 instrument.10 We demonstrated that the extracted viral RNA, which is extremely sensitive to degradation in non-extracted form, is stable at ambient temperature for three days. Here, we will adapt our ESP device to isolate viral RNA from wastewater, such that stabilized RNA may be transported to a central lab for quantitation via RT-qPCR. This technology will support environmental monitoring of SARS-CoV2 in communities without wastewater treatment facilities and building-level monitoring in high resource settings. Approach: Our objectives are to develop technology that simplifies the process for capturing SARS-CoV2 RNA from aqueous environmental sources to support wastewater and environmental surveillance while also performing fundamental measurements on SARS-CoV2 RNA stability in order to better model technology implementation for future field studies. We will achieve our objectives via the following two Specific Aims: Specific Aim 1: Simplify RNA extraction from wastewater and environmental surface water using ESP. The primary difference between our prior HIV RNA work and the proposed project is the type and scale of the sample. Wastewater systems are dilutive in nature; we expect viral RNA concentrations to be much lower in wastewater. While our HIV system accommodated a 200-500 µL blood sample, the literature suggests that 10- 200 mL samples will be necessary to detect SARS-CoV2 RNA in areas with active outbreaks2,3 (volumes are often collected using an autosampling device, which collects several small volumes of wastewater over time). The first Aim of this proposal is to develop a flow-through RNA concentration/purification device that could be exposed to a wastewater flow for minutes to hours to accumulate signal. While prior ESP configurations involved mixing magnetic beads coated with RNA capture resin, we anticipate that this strategy will lead to inefficient capture with higher sample volumes. Our approach will be to immobilize the RNA capture resin within a porous bed (e.g., a packed column of beads laden with capture resin), such that wastewater must flow through the bed, resulting in RNA accumulation. We will perform a parametric optimization of this strategy by modulating several variables including the concentration of the capture resin, the geometry of the flow-through component, and wastewater exposure time. Additionally, we will compare three RNA extraction strategies: 1 Keck, James W 1. Place the component directly in the flow of wastewater, as this will be the simplest and most direct way to interrogate the full volume of the sample. 2. Add wastewater to a circulating continuous stir bioreactor and allow flow to circulate through the component within the bioreactor to allow for the use of additives that may enhance RNA binding (e.g., salt, detergent). 3. Place an electronegative filter in a circulating continuous stir bioreactor and decrease wastewater pH to promote RNA binding to the filter and then elute onto the ESP component for purification to utilize a “tried- and-true” method of wastewater concentration but add an additional step. Following binding, ESP will be used to wash away excess wastewater, which is inhibitory to RT-PCR, and to elute the captured RNA into an RT-PCR friendly buffer. RT-qPCR will be performed using the CDC RT-qPCR assay and primer sequences.11 Overall performance of the RNA concentration/purification component will be assessed using wastewater (collected at Town Branch Wastewater Treatment Plant in Lexington, Kentucky) spiked with known quantities of SARS-CoV2 RNA (obtained from BEI Resources). We will quantify RNA via RT- qPCR and subtract any signal measured in unspiked wastewater in case the background wastewater contains SARS-CoV2. This copies-above-background measurement will be compared with the known RNA spike quantity to evaluate assay efficiency. Specific Aim 2: Measure SARS-CoV2 RNA stability under various aqueous environmental conditions Aim 2A: Little is known about the environmental stability of SARS-CoV2 viral RNA in wastewater or open water. Understanding the rate of viral RNA decay under various environmental exposures is necessary to quantify the sensitivity of the WBE approach for detecting SARS-CoV2. We will conduct a series of experiments using SARS- CoV2 RNA standardized samples from BEI Resources. We will spike wastewater and environmental (stream) water to mimic the conditions under which WBE surveillance would occur. Wastewater and environmental samples will be tested for SARS-CoV2 prior to spiking. We will expose spiked samples to a spectrum of environmental conditions including UV radiation, temperature, and pH values using a continuous stir bioreactor. We will assess RNA stability by quantifying RNA via RT-qPCR at 12, 24, 48, and 96 hours post-spiking. Aim 2B: The simplicity of ESP technology enables RNA to be extracted in nearly any setting (e.g., at the point of sample acquisition). Given that RNA is among the most labile biomarkers, the logistics of sampling in remote locations are complicated by the need to preserve the analyte, as degraded samples underestimate RNA levels. Typically, samples are packed in ice and transported to a central facility for processing,3 but cold chain shipping is difficult in many parts of the world and ice does not completely prevent RNA degradation. Fortunately, using HIV viral RNA, we have previously demonstrated that ESP-based RNA extraction can be utilized as a stabilization strategy (i.e., RNA is isolated from degrading ribonucleases). Here, we will quantify the effectiveness of this strategy for SARS-CoV2. Samples will again be prepared by spiking RNA into wastewater and samples will be stored for up to one week under different storage conditions (e.g., ambient, on ice, elevated temperature). Extracted RNA will be quantified as in Aim 1 and results will be compared against samples processed immediately after spiking RNA (positive control) and samples with ESP-stabilized RNA that were also aged up to one week. The difference between these measurements will enable us to calculate the effectiveness of our ESP-as-stabilization process. Furthermore, this accomplishment will also serve as the first step toward a complete in-the-field WBE device where RT-qPCR is replaced with simplified detection technologies (e.g., isothermal amplification methods, with which we have experience). Future Directions: While developing proof of concept in the lab we will pursue local, regional, and international partnerships for field testing of the technology. Dr. Hobbs has an existing relationship with the Town Branch Wastewater Treatment Plant in Lexington, Dr. Berry has connections with the biotech industry, and Dr. Keck serves as a consultant to the Lexington Fayette County Health Department. All three team members maintain international partnerships with sites in Latin American and Africa. We will publish research findings in relevant engineering, environmental science and public health journals. We anticipate aggressive pursuit of extramural funding once we have proof of concept. There are numerous applicable federal grant mechanisms through NIH (Environmental Health Sciences and Biomedical Imaging and Bioengineering) and NSF, including: ? NIEHS RFA-ES-19-011 R21: Time sensitive research opportunities in environmental health sciences ? NIEHS NOT-ES-20-20: Understanding the Impact of Environmental Exposures on Coronavirus Disease ? NIBIB NOT-EB-20-007: Development of Biomedical Technologies for Coronavirus Disease 2019 ? NIBIB / NIH POCTRN: Rapid Acceleration of Diagnostics (RADx) ? NSF Rapid Response Research (RAPID): https://www.nsf.gov/pubs/2020/nsf20052/nsf20052.jsp ? NSF Biosensing: https://www.nsf.gov/funding/pgm_summ.jsp?pims_id=505720 2