BACKGROUND: Single-sampling strategy (SSS) for blood cultures (BC) has not been evaluated in infective endocarditis (IE). We assessed the diagnostic performance of SSS vs conventional multi-sampling strategy (MSS) in IE diagnosis. METHODS: Patients suspected of IE were prospectively enrolled in 8 tertiary-care centers. Five BC bottle pairs were sampled from each patient. Pairs 1, 2 and 3 were sampled simultaneously, followed by 2 additional separate pairs (4 and 5) sampled more than one hour later. Pairs 1, 2, and 3 emulated SSS and pairs 1, 4, and 5 emulated MSS. The sensitivity and specificity of the major microbiologic criterion of the 2015-ESC IE diagnostic criteria, based on the SSS and MSS BC results, were calculated using the endocarditis team's diagnosis as the gold standard. RESULTS: An IE was diagnosed in 101 (39.4%) of the 256 patients enrolled. Sensitivity rates of SSS and MSS were 50.5% (95%CI [40.7-60.2]) and 45.5% (95%CI [35.8-55.3]), respectively (p = 0.063), while specificity rates were 94.8% (95%CI [91.4-98.3]) and 95.5% (95%CI [92.2-98.8]), respectively (p = 1). In IE patients, SSS as compared to MSS accurately upgraded the diagnosis from possible to definite IE in one patient and downgraded it in none. CONCLUSION: Using SSS to define the major microbiologic criterion was as sensitive and specific as using MSS for diagnosing IE. Using SSS instead of MSS BC results did not lead to erroneous changes in diagnostic class according to the 2015 ESC criteria. Consequently, SSS may be regarded as standard practice for IE diagnosis.< Réduire