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Aetiological investigations of pericardial effusion are often negative. We evaluate the interest of systematic analysis of fluid in order to diminish the number of pericarditis classified as idiopathic.We performed a systematic analysis of pericardial fluid and biopsy specimens, using cultures and molecular analyses for the identification of bacteriological, fungal, and viral agents, as well as histopathological examination of 106 pericardial fluid samples. The aetiological diagnosis was determined clinically and by non-invasive procedures in 40 and nine patients, respectively. In the remaining 57 patients, 14 neoplasias and 17 infections were diagnosed. Molecular procedures identified seven viral (Enterovirus, Herpes simplex virus, and Epstein–Barr virus in four, two, and one of the cases, respectively), one fungal (Cryptococcus neoformans), and nine bacterial infections. Four of these bacteria were not diagnosed by culture because of prior antibiotics treatment (Mycobacterium tuberculosis in two cases, Streptococcus pneumoniae in one case, and Actinomyces neuii in one case). The aetiology remained undetermined in 26 patients.The systematic use of molecular techniques permitted a significant increase in aetiological diagnoses of punctured pericardial effusions when compared with cultures (39.5 vs. 13.9%, respectively; P<0.01). It is particularly beneficial for patients with a previous antibiotic regimen or suspicion of tuberculosis.