SIGNIFICANCE (Research Strategy)

Although COVID-19 is spread primarily through direct person-to-person airborne routes, studies have shown that the RNA of SARS-CoV-2 is found in feces and in sanitary sewage (i.e., wastewater) (22, 47, 65), with shedding occurring from some pre-symptomatic and asymptomatic individuals at levels of 102 to 107 genomic copies (gc)/ml in feces and 102 to 105 gc/ml in urine (24). This is very significant, as measurement of SARS-CoV-2 RNA in wastewater can serve as an early warning of an impending outbreak (3, 31, 32, 33, 40), usually about a week (42) or two (49) before human surveillance systems are able to detect infection among the population. As a result, measurement of infectious agents in wastewater through wastewater-based testing (WBT) can empower communities by providing early warnings of outbreaks (34, 38, 56), which can be a key factor in combating the spread of any disease, including COVID-19.

In addition to serving as an early warning system for disease transmission, advantages of wastewater based testing (WBT) include accounting for individuals who are underestimated by clinical surveillance (8, 28, 55, 63, 68) because they have not pursued testing, many of which may be asymptomatic or expressing only mild symptoms (35, 38). WBT ties infection to a building and not to a person, thus overcoming the stigma of having individuals identified as transmitting the illness, which may cause some individuals to avoid seeking testing. Thus, wastewater viral concentrations represent the collective shedding by the community at a point in time, providing a temporal distribution of SARS-CoV-2 infection that requires fewer measurements than human surveillance systems. Due to these advantages, WBT for SARS-CoV-2 has been implemented throughout the world as part of wastewater-based epidemiologic (WBE) efforts aimed at using wastewater detection methods to track infection among communities (12, 19, 52).

Although there are many advantages of WBT, its use and implementation need optimization. This includes establishing better maps between WBT measures and human surveillance systems, improvements in detection sensitivity and quantification, and better strategies for sample collection, concentration, and detection (26). Wastewater is highly variable in nature and therefore there is a need to identify appropriate sample collection strategies that balance the need for broadly representative samples with the need for effective targeted virus detection, given the decay rates of the virus in samples. In general, detection in wastewater occurs when confirmed cases reach 1 to 100 per million population (4, 42, 64), and the variability in viral detection limits can be addressed by establishing consistent sample concentration and detection strategies. Efforts are needed to further decrease detection limits, increase speed, and establish standardization, enabling comparisons among communities.

A significant impact of the proposed research will be that it compares SARS-CoV-2 human surveillance data to WBT measurements of SARS-CoV-2, non-COVID microbes, and physical-chemical parameters, to improve sensitivity and specificity of wastewater measurements. Results will be used to standardize sample collection and concentration procedures. Innovative methods of sample concentration and detection will be explored to provide for more rapid processing and lower limits of detection, while at the same time providing for simplified detection procedures. Results of measurements will be used to develop a model that predicts local and community spread of COVID-19 based upon WBT. Thus, the results of the proposed research will broaden the scope of communities able to implement WBT to augment human-disease surveillance programs. Moreover, the results will inform and improve public health and safety strategies at the local community and municipal levels and may also impact public health strategies at the state, national, and international levels.

INNOVATION

Innovation is integrated throughout this proposal through the detailed human surveillance information that can be compared and correlated with WBT results for the RNA of SARS-CoV-2, and through the unique aspects of the proposed wastewater testing program aimed to optimize relationships between human surveillance results and wastewater sampling, concentration, and detection strategies.

Integration of SARS-CoV-2 WBT with COVID-19 Community Surveillance

One of the unique aspects of this proposed study is that the WBT results will be integrated with one of the first and currently still one of the most extensive community COVID-19 surveillance efforts in the US. Human surveillance for COVID-19 is ongoing within the WBT study area of the county of Miami-Dade, Florida, and at the community-level within the UM campuses. Miami-Dade County (1.2 million acres in land area) houses a diverse population of 2.72 million people (57). Wastewater is treated within the county at three major wastewater treatment plants (WWTP), the largest of which is the Central District (CD) WWTP, which treats 6.3 m3/s (143 million gallons per day) of sewage, providing sewer services for a population of about 1.1 million people. At the county level, patient testing (as of Sept. 6, 2020) has been facilitated through nine drive-through testing sites, eight walk-up testing sites, six homebound testing centers, two municipal testing sites, plus 16 additional commercial sites. Results from patient testing are reported to the Florida Department of Health for posting on their dashboard (15, floridahealthcovid19.gov), on a county and zip-code basis, and with daily case documentation by demographics (age, gender, race, ethnicity, resident, non-resident, etc.). Data is also consolidated by the County and reported daily on the internet in a simple report format (43, miamidade.gov/information/library/2020-09-05-new-normal-dashboard.pdf).

County-wide patient testing was augmented by a surveillance program (led by team members Drs. Kobetz and Solle) by randomly testing 750 people per week over a set period in April 2020, in an effort to better quantify the percentage of the population that is actually infected (not just those seeking a test). Results of the surveillance program indicated that 6% of the sample presented with COVID-19 antibodies, of which nearly 60% of the cases reported to be asymptomatic. The extent of testing and documentation of cases in Miami-Dade County, including UM led county surveillance programs provides a unique opportunity to match human surveillance data at the county level with the results from WBT from samples collected at the county-level wastewater treatment plant.

Human surveillance at the community-level has been facilitated at the UM campuses. UM is a private research university with a population of more than 34,000 students, faculty, and staff. We have access to all three primary campuses: 1) Coral Gables campus (239 acres), which includes most of the teaching and dormitory facilities for the undergraduate population; 2) Rosenstiel School of Marine and Atmospheric Science (RSMAS) campus, which houses predominantly research and administrative buildings that focus on studies of marine systems; and 3) Miller School of Medicine (MSoM) campus, which houses research and administrative facilities plus the University’s medical school, the Sylvester Comprehensive Cancer Center (Sylvester), and three University-owned hospitals. One of these hospitals (UM Hospital, UMH) treats almost all related COVID-19 patients.

UM has made tremendous efforts in adapting and responding to the unprecedented events of COVID-19. The University of Miami Health System (UHealth) rapidly built a robust SARS-CoV-2 testing program (45a); members of our research team (Williams, Shukla, and Kobetz) were leaders in this effort. Members of our research team (Kobetz and Solle) are also leading the University wide human surveillance program through a Testing, Tracing and Tracking (3-T) program. This ongoing program is essential for identifying any uptick in infection and for responding effectively and efficiently for mitigation. The surveillance initiative aims to monitor changes in infection burden that may require a different public health strategy or response for the UM community.

The surveillance program includes almost all UM students and selected faculty and staff. The UM community are being screened for COVID-19 via mid-nasal PCR testing on a weekly basis. Our team will have full access to daily test results as well as the housing locations of the residential students and work areas of the faculty and staff, to link with the wastewater samples collected throughout the three campuses. Each week, a total of 3,800 residential students are being screened in person via mid-nasal, observational PCR testing. Approximately 9,000 commuter students are being be tested weekly via home-based PCR tests and our team will have access to all results. In addition to the students, a random sample of 100 faculty and staff members are being screened in person via mid-nasal, observational PCR testing. The simple random sample selection is based on equal probability of being selected for qRT-PCR COVID-19 testing surveillance based on faculty or staff designation. This property of probability sampling avoids selection bias and enables us to use statistical theory to make valid inferences from the sample to survey the faculty population for COVID-19 testing. Results are evaluated to identify the number of new cases, where those cases are concentrated and whether they are linked. Information about the number of cases is posted on the University’s COVID-19 dashboard (UM 2020, https://coronavirus.miami.edu/dashboard). Data is interpreted to identify “hot spots” in the community and to establish potential mitigation measures (isolation and/or quarantine) for affected groups of individuals. The human surveillance program was submitted to the University’s Internal Review Board (IRB), where it was deemed not human subjects research, based on Office for Human Research Protections (OHRP) guidelines. As we move forward, we will continue to communicate with the IRB to ensure that we are in full compliance and the safety of participants is the highest priority.

Human surveillance samples collected through the UM 3-T program are processed at the UM Mailman Center while clinical patient samples are processed at the UMH Core Laboratory. Surveillance testing for faculty and staff is primarily done through nasal sampling. Samples are placed in viral transport media or universal transport media. They are processed using the PerkinElmer assay and extraction is done with Chemagic. qRT-PCR is completed using QuantStudio 7 or Applied Biosystems 7500 Fast. For symptomatic employees and patients, nasal samples are placed in the solutions described and processed using one of the following automated devices: DiaSorin, Mesa Accula, BD Max, or GenMark Diagnostics. Previously biobanked human samples collected through this COVID-19 tracking effort, for which we have permission to include in this study, will be analyzed using the same molecular detection assays as for the wastewater samples collected through this proposed WBT study. These assays include qRT-PCR, qLAMP, FA, and metatranscriptomic analyses. The fact that UM will process both human-derived and environmental COVID-19 related samples will provide a unique opportunity to match the molecular signatures from human surveillance with wastewater data.

Results from the proposed WBT study will inform and improve the University’s health and safety strategies, including student housing planning and availability of designated quarantine living spaces for students. Findings from the daily human surveillance program will be compared with the wastewater estimation of prevalence; intervention efforts will be based on this interaction. All WBT results will be QC’ed, shared with University leadership, and then presented on the publicly accessible UM COVID Dashboard, reporting daily results, trends and hotspots on each of the three University campuses. Hot spots will be distinguished on the UM Dashboard through heat maps and data will be shared with the UM community through UM’s online COVID education and communication portal.