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Background: Key indicators of vulnerability for the syndemic of opioid overdose, human immunodeficiency virus (HIV), and hepatitis C virus (HCV) due to injection drug use (IDU) in rural reservation and frontier counties are unknown. We examined county-level vulnerability for this syndemic in South Dakota.Methods: Informed by prior methodology from the Centers for Disease Control and Prevention, we used acute and chronic HCV infections among persons aged ≤40 years as a proxy measure of IDU. Twenty-nine county-level indicators potentially associated with HCV infection rates were identified. Using these indicators, we examined relationships through bivariate and multivariate analysis and calculated a composite index score to identify the most vulnerable counties (top 20%) to this syndemic.Results: Of the most vulnerable counties, 69% are reservation counties and 62% are rural. The county-level HCV infection rate is 4 times higher in minority counties than nonminority counties, and almost all significant indicators of opioid-related vulnerability in our analysis are structural and potentially modifiable through public health interventions and policies.Conclusions: Our assessment gives context to the magnitude of this syndemic in rural reservation and frontier counties and should inform the strategic allocation of prevention and intervention services. [ABSTRACT FROM AUTHOR]