ABSTRACT COVID-19 continues to create significant challenges for individuals with opioid use disorder (OUD). During the pandemic, opioid overdose rates increased by more than 50% in 37 of 38 US jurisdictions and emergency medical service calls for opioid related overdose have increased by 50%. This increase in calls has been mirrored by a 70% increase in refusal to transport rates. Opioid related morbidity, especially retention and adherence to medication assisted treatment (MAT) and relapse are increasingly difficult to measure given decreased overall interaction of individuals with OUD with the healthcare system and relaxed rules around buprenorphine and methadone prescribing and waivers of in-person drug testing due to expanded telehealth capabilities. Wastewater-based epidemiology is a feasible, noninvasive method to measure both the extent of COVID-19 and the prevalence of MAT. Analysis of wastewater both upstream in neighborhoods and downstream at wastewater treatment plants has been demonstrated as a feasible method to conduct continuous surveillance of the penetrance of COVID-19 and opioid consumption. By measuring trends in wastewater over time, insights into the effects of the burden of COVID-19 can be mapped in conjunction with drugs of abuse, thereby permitting a longitudinal assessment of the ongoing effects of COVID-19 on OUD. This urgent competitive revision expands our work in the parent grant where we have developed an innovative method to measure and visualize opioids and their key metabolites in wastewater networks. In the first phase of the parent grant, we optimized a wastewater-based opioid assay that can be applied both to wastewater treatment plants as well as community level manholes. We also developed best practices around sample collection methodology to account for population migrations and deliver a commercial opioid analysis product for cities. During the COVID-19 pandemic, we have separately developed a SARS-CoV-2 wastewater assay and modeled the ability of wastewater-based COVID-19 prevalence estimates to foreshadow rises in clinical cases and hospitalizations. This urgent competitive revision will integrate COVID-19 wastewater testing into the Biobot Analytics portfolio of commercial wastewater assay products. By innovatively developing standard operating procedures to measure SARS-CoV-2 as well as opioid use (MAT and illicit opioids) in wastewater networks, we will demonstrate the feasibility of developing maps and dashboards that lend empiric insight into the intersection of OUD, MAT and COVID-19. Our research will enhance public health efforts at monitoring the opioid epidemic and COVID pandemic at the community level by developing sustainable techniques that measure use of MAT in the context of fluctuating COVID-19 disease burden in vulnerable individuals with OUD. OMB No. 0925-0001/0002 (Rev. 03/16 Approved Through 10/31/2018) Page Continuation Format Page

Project Narrative The COVID-19 pandemic continues to disproportionately affect individuals with opioid use disorder (OUD). The use of wastewater analysis can lend population-level insights into the effects of COVID-19 on buprenorphine and methadone use as well as community-level illicit opioid use. Developing the ability to simultaneously assay wastewater for SARS-CoV-2 as well as opioids can help public health officials, researchers and community leaders visualize opioid use in the context of COVID-19.