Hubert Lepidi

Risk of Embolism and Death in Infective Endocarditis: Prognostic Value of Echocardiography. A Prospective Multicenter Study

Background—The incidence of embolic events (EE) and death is still high in patients with infective endocarditis (IE), and data about predictors of these 2 major complications are conflicting. Moreover, the exact role of echocardiography in risk stratification is not well defined. Methods and Results—In a multicenter prospective European study, including 384 consecutive patients (aged 57±17 years) with definite IE according to Duke University criteria, we tested clinical, microbiological, and echocardiographic data as potential predictors of EE and 1-year mortality. Transesophageal echocardiography was performed in all patients. Embolism occurred before or after IE diagnosis (total-EE) in 131 patients (34.1%) and after initiation of antibiotic therapy (new-EE) in 28 patients (7.3%). Staphylococcus aureus and Streptococcus bovis were independently associated with total-EE, whereas vegetation length >10 mm and severe vegetation mobility were predictors of new-EE, even after adjustment for S aureus and S bovis. One-year mortality was 20.6%. In multivariable analysis, independently of the other predictors of death (age, female sex, creatinine serum >2 mg/L, moderate or severe congestive heart failure, and S aureus) and comorbidity, vegetation length >15 mm was a predictor of 1-year mortality (adjusted relative risk=1.8; 95% CI, 1.10 to 2.82; P=0.02). Conclusions—In IE, vegetation length is a strong predictor of new-EE and mortality. In combination with clinical and microbiological findings, echocardiography may identify high-risk patients who will need a more aggressive therapeutic strategy.

Cultivation of the bacillus of Whipple's disease

BACKGROUND Whipples disease is a systemic bacterial infection, but to date no isolate of the bacterium has been established in subculture, and no strain of this bacterium has been available for study. METHODS Using specimens from the aortic [corrected] valve of a patient with endocarditis due to Whipples disease, we isolated and propagated a bacterium by inoculation in a human fibroblast cell line (HEL) with the use of a shell-vial assay. We tested serum samples from our patient, other patients with Whipples disease, and control subjects for the presence of antibodies to this bacterium. RESULTS The bacterium of Whipples disease was grown successfully in HEL cells, and we established subcultures of the isolate. Indirect immunofluorescence assays showed that the patients serum reacted specifically against the bacterium. Seven of 9 serum samples from patients with Whipples disease had IgM antibody titers of 1:50 or more, as compared with 3 of 40 samples from the control subjects (P<0.001). Polyclonal antibodies against the bacterium were generated by inoculation of the microorganism into mice and were used to detect bacteria in the excised cardiac tissue from our patient on immunohistochemical analysis. The 16S ribosomal RNA gene of the cultured bacterium was identical to the sequence for Tropheryma whippelii identified previously in tissue samples from patients with Whipples disease. The strain we have grown is available in the French National Collection. CONCLUSIONS We cultivated the bacterium of Whipples disease, detected specific antibodies in tissue from the source patient, and generated specific antibodies in mice to be used in the immunodetection of the microorganism in tissues. The development of a serologic test for Whipples disease may now be possible.

Comprehensive Diagnostic Strategy for Blood Culture-Negative Endocarditis: A Prospective Study of 819 New Cases

BACKGROUND. Blood culture-negative endocarditis (BCNE) may account for up to 31% of all cases of endocarditis. METHODS. We used a prospective, multimodal strategy incorporating serological, molecular, and histopathological assays to investigate specimens from 819 patients suspected of having BCNE. RESULTS. Diagnosis of endocarditis was first ruled out for 60 patients. Among 759 patients with BCNE, a causative microorganism was identified in 62.7%, and a noninfective etiology in 2.5%. Blood was the most useful specimen, providing a diagnosis for 47.7% of patients by serological analysis (mainly Q fever and Bartonella infections). Broad-range polymerase chain reaction (PCR) of blood and Bartonella-specific Western blot methods diagnosed 7 additional cases. PCR of valvular biopsies identified 109 more etiologies, mostly streptococci, Tropheryma whipplei, Bartonella species, and fungi. Primer extension enrichment reaction and autoimmunohistochemistry identified a microorganism in 5 additional patients. No virus or Chlamydia species were detected. A noninfective cause of endocarditis, particularly neoplasic or autoimmune disease, was determined by histological analysis or by searching for antinuclear antibodies in 19 (2.5%) of the patients. Our diagnostic strategy proved useful and sensitive for BCNE workup. CONCLUSIONS. We highlight the major role of zoonotic agents and the underestimated role of noninfective diseases in BCNE. We propose serological analysis for Coxiella burnetii and Bartonella species, detection of antinuclear antibodies and rheumatoid factor as first-line tests, followed by specific PCR assays for T. whipplei, Bartonella species, and fungi in blood. Broad-spectrum 16S and 18S ribosomal RNA PCR may be performed on valvular biopsies, when available.

Molecular Diagnosis of Infective Endocarditis by PCR Amplification and Direct Sequencing of DNA from Valve Tissue

ABSTRACT 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.

PCR Detection of Bacteria on Cardiac Valves of Patients with Treated Bacterial Endocarditis

ABSTRACT We used broad-range PCR amplification and sequencing to detect and identify bacterial DNA in 156 valves of patients treated for infective endocarditis (IE). Bacterial DNA was found more frequently in patients who underwent valve replacement while on antibiotic treatment for IE (60%) than in patients who had completed antibiotic treatment for IE (37%; P = 0.02). We found specific bacterial DNA in valves removed from 11 of 30 patients who had completed antibiotic treatment for IE. Six had no histological evidence of IE. The presence of DNA was significantly correlated with the presence of histologic lesions (P = 0.001) and with the presence of bacteria detected by Gram staining (P < 0.001). Bartonella and streptococci were detected for much longer after antibiotic treatment by PCR than other species (P = 0.047 and 0.04, respectively), and coagulase-negative staphylococci were detected for much shorter periods (P = 0.02). The finding that bacterial DNA was more likely to be detected in valves of patients with active IE than in patients who had completed antibiotic treatment for IE shows that bacterial DNA is cleared slowly. There was no significant correlation between the duration of antibiotic therapy and the presence of bacterial DNA in valves. Since the persistence of bacterial DNA in valves does not necessarily indicate the persistence of viable bacteria, the detection of bacterial DNA in valves from IE patients should be interpreted with caution, in particular in those patients with a past history of treated IE.

Effect of Sex on Coxiella burnetii Infection: Protective Role of 17β-Estradiol

Q fever is a zoonosis caused by Coxiella burnetii and recently has been recognized as a potential agent of bioterrorism. In Q fever, men are symptomatic more often than women, despite equal seroprevalence. We hypothesized that sex hormones play a role in the pathogenesis of C. burnetii infection. When C57/BL6 mice were injected with C. burnetii, bacteria load and granuloma numbers were lower in females than in males. Ovarectomized mice showed increased bacteria load in the spleen and the liver, similar to that found in males. The granuloma number was also increased in ovarectomized mice and reached the levels found in males. Tissue infection and granulomatous response are largely under the control of estrogens: treatment of ovarectomized mice with 17beta-estradiol reduced both bacteria loads and granuloma numbers. These results show that sex hormones control host response to C. burnetii infection and may account for host-dependent clinical presentation of Q fever.

Systemic Tropheryma whipplei: clinical presentation of 142 patients with infections diagnosed or confirmed in a reference center.

Culture of Tropheryma whipplei, the agent of Whipple disease (WD), was achieved in our laboratory in 2000, allowing new perspectives for the diagnosis of this disease and for the description of other potential clinical manifestations caused by this microorganism. Since 2000, we have developed new tools in our center in Marseille, France, to optimize the diagnosis of T whipplei infections. Classic WD was characterized by positive periodic acid-Schiff performed on duodenal biopsy. In the absence of duodenal histologic involvement, localized infections were defined by specific positive T whipplei polymerase chain reaction (PCR) results obtained using samples of other tissues and body fluids. The physicians in charge of patients were asked to complete a questionnaire. A total of 215 diagnoses were performed or confirmed and, among these, 142 patients with sufficient clinical data were included. Herein, we report epidemiologic data, clinical manifestations, and diagnostic tools of T whipplei infections. In the 113 patients with classic WD, the main symptom was arthralgia (88/113, 78%), which explains the many cases misdiagnosed as inflammatory rheumatoid disease (56/113, 50%). Frequently immunosuppressive treatments, more recently including tumor necrosis factor inhibitor, had been previously prescribed (50%) and were often responsible for more rapid clinical progression (43%). Sometimes a short course of antibiotics improved the clinical status. Endocarditis was the second most frequent manifestation of T whipplei, with 16 cases. The clinical picture of this entity corresponds to cardiovascular involvement with acute heart failure (50%) occurring without fever (75%) or previous valvular disease (69%). Neurologic symptoms were the third major manifestation. Other localized infections such as adenopathy, uveitis, pulmonary involvement, or joint involvement were sporadic. Infection with T whipplei resulted in multifaceted conditions. Some localized infections due to this agent have recently been reported and may correspond to emerging entities. Patients with inflammatory rheumatoid disease must be systematically interviewed to determine the efficacy of previous immunosuppressive and antibiotic therapies. Abbreviations: CSF = cerebrospinal fluid, PAS = periodic acid-Schiff, PCR = polymerase chain reaction, WD = Whipple disease.

Cardiac Valves in Patients with Q Fever Endocarditis: Microbiological, Molecular, and Histologic Studies

The pathologic features of Q fever endocarditis, which is caused by Coxiella burnetii, were histologically evaluated in cardiac valves from 28 patients. We used quantitative image analysis to compare valvular fibrosis, calcifications, vegetations, inflammation, and vascularization due to Q fever endocarditis with that due to non-Q fever endocarditis and valvular degeneration. We also studied the presence of C. burnetii in valves by immunohistochemical analysis, culture, and polymerase chain reaction (PCR). Histologically, Q fever endocarditis was characterized by significant fibrosis and calcifications, slight inflammation and vascularization, and small or absent vegetations. Despite antibiotic treatment, non-statistically significant variations at the histologic level were observed. These pathologic features could be confused with noninfectious valvular degenerative damage. We found that the detection of C. burnetii in cardiac valves by immunohistochemical analysis, culture, and PCR decreased significantly only after 1 year of antibiotic treatment, which emphasizes the long persistence of this organism in valve tissues. Pathologic and immunohistochemical analyses may contribute to the diagnosis of Q fever endocarditis.

Lipopolysaccharide from Coxiella burnetii Is Involved in Bacterial Phagocytosis, Filamentous Actin Reorganization, and Inflammatory Responses through Toll-Like Receptor 4

The role of Toll-like receptors (TLRs) in the recognition of extracellular and facultative intracellular bacteria by the innate immune system has been extensively studied, but their role in the recognition of obligate intracellular organisms remains unknown. Coxiella burnetii, the agent of Q fever, is an obligate intracellular bacterium that specifically inhabits monocytes/macrophages. We showed in this study that C. burnetii LPS is involved in the uptake of virulent organisms by macrophages but not in that of avirulent variants. The uptake of virulent organisms was dependent on TLR4 because it was reduced in macrophages from TLR4−/− mice. In addition, LPS was responsible for filamentous actin reorganization induced by virulent C. burnetii, which was prevented in TLR4−/− macrophages. In contrast, the intracellular fate of C. burnetii was not affected in TLR4−/− macrophages, suggesting that TLR4 does not control the maturation of C. burnetii phagosome and the microbicidal activity of macrophages. These results are consistent with in vivo experiments because the pattern of tissue infection and the clearance of C. burnetii were similar in wild-type and TLR4−/− mice. We also showed that the number of granulomas was decreased in the liver of infected TLR4−/− mice, and the formation of splenic granulomas was only transient. The impaired formation of granulomas was associated with decreased production of IFN-γ and TNF. Taken together, these results demonstrate that TLR4 controls early events of C. burnetii infection such as macrophage phagocytosis, granuloma formation, and cytokine production.

Anti-inflammatory properties of molecular hydrogen: investigation on parasite-induced liver inflammation

Molecular hydrogen reacts with the hydroxyl radical, a highly cytotoxic species produced in inflamed tissues. It has been suggested therefore to use gaseous hydrogen in a new anti-inflammatory strategy. We tested this idea, with the aid of the equipment and skills of COMEX SA in Marseille, a group who experiments with oxygen-hydrogen breathing mixtures for professional deep-sea diving. The model used was schistosomiasis-associated chronic liver inflammation. Infected animals stayed 2 weeks in an hyperbaric chamber in a normal atmosphere supplemented with 0.7 MPa hydrogen. The treatment had significant protective effects towards liver injury, namely decreased fibrosis, improvement of hemodynamics, increased NOSII activity, increased antioxidant enzyme activity, decreased lipid peroxide levels and decreased circulating TNF-alpha levels. Under the same conditions, helium exerted also some protective effects, indicating that hydroxyl radical scavenging is not the only protective mechanism. These findings indicate that the proposed anti-inflammatory strategy deserves further attention.