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Despite progress with diagnostic criteria, the type and timing of laboratory tests used to diagnose infective endocarditis (IE) have not been standardized. This is especially true with serological testing. Patients with suspected IE were evaluated by a standard diagnostic protocol. This protocol mandated an evaluation of the patients according to the modified Duke criteria and used a battery of laboratory investigations, including three sets of blood cultures and systematic serological testing for Coxiella burnetii , Bartonella spp., Aspergillus spp., Legionella pneumophila , and rheumatoid factor. In addition, cardiac valvular materials obtained at surgery were subjected to a comprehensive diagnostic evaluation, including PCR aimed at documenting the presence of fastidious organisms. The study included 1,998 suspected cases of IE seen over a 9-year period from April 1994 to December 2004 in Marseilles, France. They were evaluated prospectively. A total of 427 (21.4%) patients were diagnosed as having definite endocarditis. Possible endocarditis was diagnosed in 261 (13%) cases. The etiologic diagnosis was established in 397 (93%) cases by blood cultures, serological tests, and examination of the materials obtained from cardiac valves, respectively, in 348 (81.5%), 34 (8%), and 15 (3.5%) definite cases of IE. Concomitant infection with streptococci and C. burnetii was seen in two cases. The results of serological and rheumatoid factor evaluation reclassified 38 (8.9%) possible cases of IE as definite cases. Systematic serological testing improved the performance of the modified Duke criteria and was instrumental in establishing the etiologic diagnosis in 8% (34/427) cases of IE.