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Blood culture negative infective endocarditis (BCNIE) poses both a diagnostic and therapeutic challenge. High rates of BCNIE reported in South Africa have been attributed to antibiotic use prior to blood culture sampling. To assess the impact of a systematic approach to organism detection and identify the causes of infective endocarditis (IE), in particular causes of BCNIE. Prospective cohort study. The Tygerberg Endocarditis Cohort study prospectively enrolled patients with IE between November 2019 and February 2021. A set protocol fo