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In an attempt to elucidate better the various aetiologies of pericardial effusion, we developed a diagnostic protocol that incorporated a battery of systematic tests including blood cultures, throat swab cultures and serological tests for various infectious agents and estimation of serum antinuclear antibodies and serum thyroid-stimulating hormone. Over a 2-y period ending May 2000, we evaluated prospectively and diagnostic usefulness of our strategy in a cohort (n = 136) of patients with pericardial effusion treated at Hospital Timone (HT), Marseille. We compared our findings with those observed in a retrospectively (May 1998-May 2000) drawn cohort (n = 127) of patients treated at Hospital Louis Pradel (HLP), Lyon and in which the laboratory investigation towards establishing an aetiological diagnosis was undertaken intuitively. Overall, the aetiologies were obvious clinically in 18% of cases. In other cases, specific aetiologies (27.3% vs 3.9%; p < 0.001), including treatable conditions (11.1% vs 2.4%; p < 0.001) were identified significantly more frequently in the HT cohort compared to the HLP cohort. The diagnosis strategy we propose may be helpful in elucidating the aetiological diagnosis of pericardial effusion when a cause is not obvious clinically.

The pathological features of Whipple endocarditis, which is caused by Tropheryma whipplei, were histologically evaluated in cardiac valves from 5 patients. We used quantitative image analysis to compare the valvular fibrosis, calcifications, vegetations, inflammation, and vascularization due to Whipple endocarditis with those due to non-Whipple endocarditis and degenerative valves. We also studied the presence of T. whipplei in valves by immunohistochemical analysis, culture, and polymerase chain reaction (PCR). In histologic analysis, Whipple endocarditis was characterized by significant fibrosis, a lack of calcifications, slight inflammation and vascularization, and vegetations of intermediate size. Inflammatory infiltrates consisted mainly of foamy macrophages and lymphocytes. We found that the detection of T. whipplei in cardiac valves, by immunohistochemical analysis, was correlated with the detection of the bacterium by culture and PCR. We report, for the first time, the immunodetection of T. whipplei in a surgically removed arterial embolus. Pathological and immunohistologic analyses may contribute to the diagnosis of Whipple endocarditis.

We describe 2 patients with endocarditis for whom blood cultures and cardiac valve cultures were repeatedly sterile. Broad-range eubacterial polymerase chain reaction (PCR) amplification performed on cardiac valve specimens from these 2 patients detected DNA of Mycoplasma hominis, for one patient, and of Ureaplasma parvum, for the other patient. Three other cases of infective endocarditis caused by mycoplasmas were identified in the literature. It is important to rule out a diagnosis of mycoplasma endocarditis because the evolution of the disease may be fatal and it requires an adequate and specific antibiotic therapy.

Panton Valentine leukocidin (PVL) may be responsible for pulmonary necrosis in necrotizing Staphylococcus aureus pneumonia, a highly lethal infection. Commercial intravenous immunoglobulin (IVIg) preparations containing antibodies against PVL might have therapeutic value in this setting, as an adjunct to antimicrobial chemotherapy. To test this possibility, we determined anti-PVL antibody titers in commercial IVIg and the capacity of IVIg to prevent the cytopathic effects of PVL in vitro. Specific enzyme-linked immunosorbent assays based on purified recombinant PVL (rPVL) showed that IVIg contained specific anti-PVL antibodies. The cytotoxicity of rPVL and of crude culture supernatants of PVL-producing S. aureus strains were investigated by measuring ethidium-bromide incorporation by polymorphonuclear neutrophils (PMNs) in flow cytometric assays, as well as PMN ultrastructural changes by transmission electron microscopy. IVIg was found to neutralize pore formation and the cytopathic effect of both rPVL and S. aureus culture supernatants.

The culture of Tropheryma whipplei, the bacterium responsible for Whipple's disease, has been established only recently. Our objective is to describe, based on our experience, the culture of T. whipplei in HEL cells detected by immunofluorescence staining. Over 3 years, we received 18 samples for T. whipplei culture from 15 patients with Whipple's disease. Ten duodenal biopsy specimens from 10 patients with digestive symptoms were available. Five cardiac valves and three blood samples from five patients with endocarditis were also available. We correlated the results of culture with the type of sample and the culture procedure. Seven isolates were obtained, and three were subsequently established for more than 4 passages. The mean delay for the primary detection was 30 days. The bacterium was isolated more frequently from sterile specimens (5 of 8) than from duodenal biopsy specimens (2 of 10), but the difference (P = 0.14) was not significant. Decontamination of digestive samples containing colistin, amphotericin B, and cephalotin or ciprofloxacin did not impair the isolation of T. whipplei. The use of vancomycin precludes the primary isolation (7 of 12 versus 0 of 6; P = 0.08) and the establishment of T. whipplei (3 of 12 versus 0 of 6; P = 0.5). Omitting samples cultured with vancomycin, the establishment of the strain was significantly higher when antibiotics were prescribed for no more than 7 days (3 of 4 versus 0 of 8; P = 0.03). Our results demonstrate that samples must be collected within 1 week of an antibiotic regimen's initiation for the successful establishment of the bacterium.

We used broad-range eubacterial PCR amplification followed by direct sequencing to identify microbial pathogens in heart valve material from 29 patients with histologically confirmed infective endocarditis and 23 patients free of infective endocarditis. Microorganisms cultured by conventional techniques matched those identified by PCR in 21 cases. PCR alone identified the causative agent in three cases (Streptococcus bovis, Staphylococcus cohnii, and Coxiella burnetii), allowing better patient management. PCR corrected the initial bacteriological diagnosis in three cases (Streptococcus bovis, Streptococcus mutans, and Bartonella henselae). Among the 29 cases of histologically confirmed infective endocarditis, PCR findings were positive in 27 cases and were consistent with the bacterial morphology seen at Gram staining (26 cases) or with the results obtained by immunohistologic analysis with an anti-C. burnetii monoclonal antibody (one case). In two other cases of histologically confirmed infective endocarditis, PCR remained negative in a blood culture-negative case for which no bacteria were seen at histological analysis and in one case with visualization of cocci and blood cultures positive for Enterococcus faecalis. Ten clinical diagnoses of possible infective endocarditis were ruled out by histopathological analysis of the valves and subsequently by PCR. PCR was negative in 13 of the 14 patients in whom infective endocarditis was rejected on clinical grounds; the other patient was found to have Coxiella burnetii infective endocarditis on the basis of PCR and histopathological analysis and was subsequently included in the group of 29 definite cases. In total, PCR contributed to the diagnosis and management of infective endocarditis in 6 of 29 (20%) cases.

The binding of the S component (LukS-PV) from the bicomponent staphylococcal Panton-Valentine leucocidin to human polymorphonuclear neutrophils (PMNs) and monocytes was determined using flow cytometry and a single-cysteine substitution mutant of LukS-PV. The mutant was engineered by replacing a glycine at position 10 with a cysteine and was labeled with a fluorescein moiety. The biological activity of the mutant was identical to that of the native protein. It has been shown that LukS-PV has a high affinity for PMNs (Kd = 0.07 +/- 0.02 nM, n = 5) and monocytes (Kd = 0.020 +/- 0.003 nM, n = 3) with maximal binding capacities of 197,000 and 80,000 LukS-PV molecules per cell, respectively. The nonspecifically bound molecules of LukS-PV do not form pores in the presence of the F component (LukF-PV) of leucocidin. LukS-PV and HlgC share the same receptor on PMNs, but the S components of other staphylococcal leukotoxins, HlgA, LukE, and LukM, do not compete with LukS-PV for its receptor. Extracellular Ca2+ at physiological concentrations (1 to 2 nM) has only a slight influence on the LukS-PV binding, in contrast to its complete inhibition by Zn2+. The down-regulation by phorbol 12-myristate 13-acetate (PMA) of the binding of LukS-PV was blocked by staurosporine, suggesting that the regulatory effect of PMA depends on protein kinase C activation. The labeled mutant form of LukS-PV has proved very useful for detailed binding studies of circulating white cells by flow cytometry. LukS-PV possesses a high specific affinity for a unique receptor on PMNs and monocytes.