Biomedical subjects
Didier Raoult
Publications and source records attributed to Didier Raoult.
Pneumonia in mice inoculated experimentally with Acanthamoeba polyphaga mimivirus.
In an attempt to elucidate whether Acanthamoeba polyphaga mimivirus (APM) was pathogenic (i.e., induces histological evidence of inflammation or tissue destruction or both) in mammalian host, we inoculated adult BALB/c (n=6) and C57BL/6 (n=6) mice with APM via intracardiac route. C57BL/6 mice developed histopathological features of pneumonia by post-inoculation day (PID) 3 and BALB/c mice, by PID7. The histopathological features of pneumonia, characterized by the presence of thickened alveolar walls with cellular infiltrates comprising mononuclear leukocytes, macrophages and lymphocytes, diffuse alveolar damages with the formation of hyaline membrane and erythrocytes in the alveolar lumen, were most marked by PID 7. We conclude that APM is pathogenic in mice in experimental conditions.
Role of sex, age, previous valve lesion, and pregnancy in the clinical expression and outcome of Q fever after a large outbreak.
BACKGROUND: Q fever is a zoonosis caused by Coxiella burnetii. After a large outbreak occurred in the Chamonix Valley in the French Alps in 2002, an extensive surveillance was conducted, to describe the variations in the clinical expression of acute Q fever according to host factors, as well as to monitor the risk of evolution of acute Q fever to chronic Q fever in patients at risk. METHODS: Three groups of patients with risk factors for evolution of acute Q fever to chronic Q fever were considered: 376 pregnant women, 19 immunocompromised patients, and 91 patients with valvular or vascular abnormalities. A group of 578 people without risk factors for evolution of acute Q fever to chronic Q fever was also tested. Diagnosis of Q fever was based on serologic testing by immunofluorescence assay. RESULTS: Between 30 August 2002 and 31 July 2003, a total of 1946 serum samples obtained from 1064 persons were tested. A total of 101 patients (9.3%) had acute Q fever diagnosed, and 5 patients (0.5%) had chronic Q fever diagnosed. A diagnosis of acute Q fever was established for 11 pregnant women (2.6% of 379 pregnancies), 5 patients with valvular disease (5.5%), and 85 people without risk factors (14.7%) (71 [27.9%] of 254 symptomatic patients and 14 [4.3%] of 324 asymptomatic patients). A new pregnancy in a woman with negative results of serologic tests for Q fever exposes the woman to a new risk for acute Q fever able to evolve to chronic Q fever. The rates of clinical expression were 90.6% in adult men, 75% in adult women, and 33.3% in children, and they were significantly lower (9.1%) in pregnant women. Evolution to chronic Q fever was observed in 5 patients. CONCLUSION: This study emphasizes the importance of active surveillance in postepidemic conditions, especially among patients at risk, as well as the importance of systematic serologic testing during pregnancy.
Experimental model to evaluate the human body louse as a vector of plague.
Yersinia pestis has been found in human body lice during plague outbreaks. To evaluate the role that the human body louse plays as a vector of plague, we allowed lice to feed on rabbits made bacteremic by intravenous inoculation of 10(9) colony-forming units of 3 strains of Y. pestis. High mortality rates were observed in all lice 2 and 3 days after infection. The lice remained infected with the strains for their life span and excreted viable organisms in their feces from day 1, although they were unable to lay eggs. The lice infected with 2 virulent strains of Y. pestis transmitted the organisms during feeding to uninfected rabbits, which became septicemic and died of plague (with 1 exception) 1 day later. Infections were transmitted to naive lice that were fed on these rabbits, showing that lice can be vectors of Y. pestis in an experimental model.
A serum protein signature with high diagnostic value in bacterial endocarditis: results from a study based on surface-enhanced laser desorption/ionization time-of-flight mass spectrometry.
BACKGROUND: Bacterial endocarditis is a serious disease. Surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS) based on serum protein profiling is a powerful approach that can generate biomarkers with diagnostic value. METHODS: To identify a protein signature associated with bacterial endocarditis, we retrospectively performed SELDI-TOF MS profiling of serum samples from 88 patients hospitalized because of clinical suspicion of endocarditis. The diagnosis was confirmed by conventional criteria for 34 patients (endocarditis positive) and was excluded for 54 patients (endocarditis negative). Serum samples were incubated with cation-exchange ProteinChip arrays. The protein profiles generated were subjected to biostatistical processing. RESULTS: Fifty-nine samples (23 endocarditis positive and 36 endocarditis negative) were randomly selected for a learning set, with the 29 remaining samples (11 endocarditis positive and 18 endocarditis negative) serving as an independent testing (validation) set. Sixty-six protein peaks were differentially expressed between the endocarditis-positive and the endocarditis-negative patients. By combining partial least squares and logistic regression methods, we built a serum protein model that perfectly discriminated between endocarditis-positive and endocarditis-negative patients. Importantly, when this model was tested on the independent testing set, a correct prediction rate of nearly 90% was demonstrated. Overall, sensitivity, specificity, positive predictive value, and negative predictive value were 94%, 98%, 96%, and 96%, respectively. CONCLUSIONS: SELDI-TOF MS profiling revealed a serum signature with high diagnostic potential for endocarditis.
Culture of T. whipplei from the stool of a patient with Whipple's disease.
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HIV-2 Protease resistance defined in yeast cells.
BACKGROUND: Inhibitors of the HIV-1 Protease currently used in therapeutic protocols, have been found to inhibit, although at higher concentrations, the HIV-2 encoded enzyme homologue. Similar to observations in HIV-1 infected individuals, therapeutic failure has also been observed for some patients infected with HIV-2 as a consequence of the emergence of viral strains resistant to the anti-retroviral molecules. In order to be able to define the specific mutations in the Protease that confer loss of susceptibility to Protease Inhibitors, we set up an experimental model system based in the expression of the viral protein in yeast. RESULTS: Our results show that the HIV-2 Protease activity kills the yeast cell, and this process can be abolished by inhibiting the viral enzyme activity. Since this inhibition is dose dependent, IC50 values can be assessed for each anti-retroviral molecule tested. We then defined the susceptibility of HIV-2 Proteases to Protease Inhibitors by comparing the IC50 values of Proteases from 7 infected individuals to those of a sensitive wild type laboratory adapted strain. CONCLUSION: This functional assay allowed us to show for the first time that the L90M substitution, present in a primary HIV-2 isolate, modifies the HIV-2 Protease susceptibility to Saquinavir but not Lopinavir. Developing a strategy based on the proposed yeast expressing system will contribute to define amino acid substitutions conferring HIV-2 Protease resistance.
Increased levels of soluble CD40L in African tick bite fever: possible involvement of TLRs in the pathogenic interaction between Rickettsia africae, endothelial cells, and platelets.
The pathophysiological hallmark of spotted fever group rickettsioses comprises infection of endothelial cells with subsequent infiltration of inflammatory cells. Based on its ability to promote inflammation and endothelial cell activation, we investigated the role of CD40L in African tick bite fever (ATBF), caused by Rickettsia africae, using different experimental approaches. Several significant findings were revealed. 1) Patients with ATBF (n = 15) had increased serum levels of soluble CD40 ligand (sCD40L), which decreased during follow-up. 2) These enhanced sCD40L levels seem to reflect both direct and indirect (through endothelial cell activation involving CX3CL1-related mechanisms) effects of R. africae on platelets. 3) In combination with sCD40L, R. africae promoted a procoagulant state in endothelial cells by up-regulating tissue factor and down-regulating thrombomodulin expression. 4) Although the R. africae-mediated activation of platelets involved TLR2, the combined procoagulant effects of R. africae and sCD40L on endothelial cells involved TLR4. 5) Doxycycline counteracted the combined procoagulant effects of R. africae and sCD40L on endothelial cells. Our findings suggest an inflammatory interaction between platelets and endothelial cells in ATBF, involving TLR-related mechanisms. This interaction, which includes additive effects between sCD40L and R. africae, may contribute to endothelial inflammation and hypercoagulation in this disorder.
Impact of the excision of an ancient repeat insertion on Rickettsia conorii guanylate kinase activity.
The genomic sequencing of Rickettsia conorii revealed a new family of Rickettsia-specific palindromic elements (RPEs) capable of in-frame insertion in preexisting open reading frames (ORFs). Many of these altered ORFs correspond to proteins with well-characterized or essential functions in other microorganisms. Previous experiments indicated that RPE-containing genes are normally transcribed and that no excision of the repeat occurs at the mRNA level. Using mass spectrometry, we now confirmed the retention of the RPE-derived amino acid residues in 4 proteins successfully expressed in Escherichia coli, raising the general question of the consequences of this common insertion event on the fitness of Rickettsia enzymes. The predicted guanylate kinase activity of the R. conorii gmk gene product was measured both on the RPE-containing and RPE-excised recombinant proteins. We show that the 2 proteins are active but exhibit substantial differences in their affinity for adenosine triphosphate, guanosine monophosphate, and catalytic constants. The distribution of the RPEgmk insert among Rickettsia species indicates that the insertion event is ancient and occurred after the divergence of Rickettsia felis and R. conorii but before that of Rickettsia helvetica and R. conorii. We found no evidence that the gmk gene fixed adaptive changes to compensate the RPE peptide insertion. Furthermore, the analysis of the rates of divergence in 23 RPE-containing genes indicates that coding RPE repeats tend to evolve under weak selective constraint, at a rate similar to intergenic noncoding RPE sequences. Altogether, these results suggest that the insertion of RPE-encoded "selfish peptides," although respecting the original fold and activity of the host proteins, might be slightly detrimental to the enzyme efficiency within limits tolerable for slow-growing intracellular parasites such as Rickettsia.
Deficient transendothelial migration of leukocytes in Q fever: the role played by interleukin-10.
Chronic Q fever is characterized by deficient cell-mediated immune response, lack of granulomas, and dysregulation of the cytokine network. Altered transendothelial migration (TM) of peripheral-blood mononuclear cells might account for impaired immune response. TM of lymphocytes and monocytes was decreased in patients with Q fever endocarditis, compared with that in patients recovering from acute Q fever and in control subjects. This defect is related to interleukin (IL)-10, a cytokine involved in the chronic evolution of the disease; neutralizing anti-IL-10 antibodies corrected TM of mononuclear cells from patients with Q fever endocarditis. IL-10 may account for deficient protective immunity in patients with Q fever endocarditis by impairing TM.
Molecular analysis of pericardial fluid: a 7-year experience.
AIMS: 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. METHODS AND RESULTS: 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. CONCLUSION: 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.
Autoimmunohistochemistry: a new method for the histologic diagnosis of infective endocarditis.
BACKGROUND: Although the pathologic examination of cardiac valves remains the reference standard for the diagnosis of infective endocarditis, the detection of microorganisms often poses a challenge for pathologists. This can be done by use of nonspecific histochemical stains or by immunohistochemical analysis, but specific antibodies are often not available. We describe a novel method for the detection of microorganisms in valve specimens from patients with infective endocarditis. METHODS: Detection of microorganisms was performed in valve specimens from patients with endocarditis caused by gram-positive cocci (25 specimens), blood culture-negative endocarditis (15 specimens: 6 cases caused by Coxiella burnetii, 5 caused by Tropheryma whipplei, and 4 caused by Bartonella species), or noninfective degenerative damage (30 specimens, used as negative controls), using the patients' own serum. This technique, called "autoimmunohistochemistry," is an immunohistochemical peroxidase-based method that we compared with results of culture and polymerase chain reaction (PCR) assay. RESULTS: Bacteria were detected by autoimmunohistochemistry in 20 (80%) specimens from patients with endocarditis caused by gram-positive cocci and in 15 (100%) specimens from patients with blood culture-negative endocarditis but in no control specimens. The rate of detection of bacteria by autoimmunohistochemistry was significantly higher than that by culture but was similar to that by PCR. CONCLUSIONS: Autoimmunohistochemistry may be useful for the detection of microorganisms in samples of valves from patients with infective endocarditis. This new diagnostic tool may be particularly useful in cases of blood culture-negative endocarditis.
Genome sequence of Rickettsia bellii illuminates the role of amoebae in gene exchanges between intracellular pathogens.
The recently sequenced Rickettsia felis genome revealed an unexpected plasmid carrying several genes usually associated with DNA transfer, suggesting that ancestral rickettsiae might have been endowed with a conjugation apparatus. Here we present the genome sequence of Rickettsia bellii, the earliest diverging species of known rickettsiae. The 1,552,076 base pair-long chromosome does not exhibit the colinearity observed between other rickettsia genomes, and encodes a complete set of putative conjugal DNA transfer genes most similar to homologues found in Protochlamydia amoebophila UWE25, an obligate symbiont of amoebae. The genome exhibits many other genes highly similar to homologues in intracellular bacteria of amoebae. We sought and observed sex pili-like cell surface appendages for R. bellii. We also found that R. bellii very efficiently multiplies in the nucleus of eukaryotic cells and survives in the phagocytic amoeba, Acanthamoeba polyphaga. These results suggest that amoeba-like ancestral protozoa could have served as a genetic "melting pot" where the ancestors of rickettsiae and other bacteria promiscuously exchanged genes, eventually leading to their adaptation to the intracellular lifestyle within eukaryotic cells.
Laboratory infection of a technician by mimivirus.
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SVARAP and aSVARAP: simple tools for quantitative analysis of nucleotide and amino acid variability and primer selection for clinical microbiology.
BACKGROUND: Simple computerized methods that analyse variability along alignments of nucleotide or amino acid sequences can be very useful in a clinical microbiology laboratory for two main purposes. First, to optimize primer selection, which is critical for the identification of infectious pathogens based on gene sequencing: primers must target conserved nucleotide regions bordering highly variable areas to ensure discrimination of species. Second, it can be of interest to reveal mutations associated with drug resistance of pathogen agents. Our aim was therefore to test easy and cost-free tools (SVARAP and aSVARAP) that require short hands-on work, little expertise, and which allow visual interpretation and statistical analysis of results. RESULTS: We first tested SVARAP to improve a strategy of identification of streptococci species of the Viridans Group targeting the groESL gene. Two regions with < 500 nucleotides were identified, one being significantly more discriminant than one of a similar length used in a previous study (mean number of nucleotide differences between species, 113 (range: 12-193) vs. 77 (range: 14-109); p < 10-3). Secondly, aSVARAP was tested on reverse transcriptase (RT) sequences from 129 HIV-1 clinical strains to identify natural polymorphisms and drug-selected mutations emerging under nucleoside RT inhibitor (NRTI)-selective pressure. It revealed eleven of the 18 RT mutations considered in a reference HIV-1 genotypic NRTI-resistance interpretation algorithm. CONCLUSION: SVARAP and aSVARAP are simple, versatile and helpful tools for analysis of sequence variability, and are currently being used in real practice in our clinical microbiology laboratory.
Identification of two putative rickettsial adhesins by proteomic analysis.
The rickettsial membrane proteins that promote their uptake by eukaryotic host cells are unknown. To identify rickettsial ligand(s) that bind host cell surface proteins, biotinylated epithelial cells were used to probe a nitrocellulose membrane containing rickettsial extracts separated by SDS-PAGE. This overlay assay revealed that two close rickettsial ligands of approximately 32-30 kDa were recognized by host cells. Both proteins were identified using high resolution 2D-PAGE coupled with mass spectrometry analysis. One protein was identified as the C-terminal extremity of rOmpB called the beta-peptide. The second interacting protein was identified as a protein of unknown function encoded by RC1281 and RP828 in Rickettsia conorii and in Rickettsia prowazekii, respectively, that shares strong similarities with other bacterial adhesins. Both proteins are highly conserved within the Rickettsia genus and might play a critical role in their pathogenicity. These data may have important implications for the development of future vaccines against rickettsial infections.
Sca1, a previously undescribed paralog from autotransporter protein-encoding genes in Rickettsia species.
BACKGROUND: Among the 17 genes encoding autotransporter proteins of the "surface cell antigen" (sca) family in the currently sequenced Rickettsia genomes, ompA, sca5 (ompB) and sca4 (gene D), have been extensively used for identification and phylogenetic purposes for Rickettsia species. However, none of these genes is present in all 20 currently validated Rickettsia species. Of the remaining 14 sca genes, sca1 is the only gene to be present in all nine sequenced Rickettsia genomes. To estimate whether the sca1 gene is present in all Rickettsia species and its usefulness as an identification and phylogenetic tool, we searched for sca1genes in the four published Rickettsia genomes and amplified and sequenced this gene in the remaining 16 validated Rickettsia species. RESULTS: Sca1 is the only one of the 17 rickettsial sca genes present in all 20 Rickettsia species. R. prowazekii and R. canadensis exhibit a split sca1 gene whereas the remaining species have a complete gene. Within the sca1 gene, we identified a 488-bp variable sequence fragment that can be amplified using a pair of conserved primers. Sequences of this fragment are specific for each Rickettsia species. The phylogenetic organization of Rickettsia species inferred from the comparison of sca1 sequences strengthens the classification based on the housekeeping gene gltA and is similar to those obtained from the analyses of ompA, sca5 and sca4, thus suggesting similar evolutionary constraints. We also observed that Sca1 protein sequences have evolved under a dual selection pressure: with the exception of typhus group rickettsiae, the amino-terminal part of the protein that encompasses the predicted passenger domain, has evolved under positive selection in rickettsiae. This suggests that the Sca1 protein interacts with the host. In contrast, the C-terminal portion containing the autotransporter domain has evolved under purifying selection. In addition, sca1 is transcribed in R. conorii, and might therefore be functional in this species. CONCLUSION: The sca1 gene, encoding an autotransporter protein that evolves under dual evolution pressure, is the only sca-family gene to be conserved by all Rickettsia species. As such, it is a valuable identification target for these bacteria, especially because rickettsial isolates can be identified by amplification and sequencing of a discriminatory gene fragment using a single primer pair. It may also be used as a phylogenetic tool. However, its current functional status remains to be determined although it was found expressed in R. conorii.
Endocarditis after acute Q fever in patients with previously undiagnosed valvulopathies.
We describe 3 cases of endocarditis after acute Q fever in 3 patients with clinically silent, undiagnosed valvulopathies, including mitral valve prolapse, minimal valvular leak, and biscuspid aortic valve. We conclude that, to prevent endocarditis, these minor valvulopathies must be actively searched for with echocardiography after diagnosis of acute Q fever.