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Didier Raoult

Publications and source records attributed to Didier Raoult.

At least 325 records · Page 18Linked to original sources
PubMed2002

Monoclonal antibodies to immunodominant epitope of Tropheryma whipplei.

Recent isolation of Tropheryma whipplei (formerly Trophyrema whippelii), the agent of Whipple's disease, from the cardiac valve of a patient with Whipple's disease endocarditis now allows the detection of reactive epitopes that could be used in a serological assay. In order to propose an enzyme-linked immunosorbent assay (ELISA) that uses recombinant T. whipplei antigen, we first determined by Western blotting of human, mouse, and rabbit antisera that the common immunodominant epitope is an 84-kDa protein. We then produced 13 monoclonal antibodies (MAbs) against T. whipplei, 12 of which recognize this immunodominant epitope. These MAbs did not react with phylogenetically closely related bacteria or bacteria previously shown to be cross-reactive with T. whipplei, but they did react with two other strains of T. whipplei isolated, one from an ocular sample and the other from a duodenal biopsy specimen. By confocal microscopy, the MAbs allowed detection of T. whipplei within infected fibroblasts. The identification of the 84-kDa antigen with our MAbs will make it possible to develop a diagnostic antigen for use in a diagnostic ELISA for Whipple's disease.

Actinobacteria↗
PubMed2002

Kinetics of antibody responses in Rickettsia africae and Rickettsia conorii infections.

African tick-bite fever, caused by Rickettsia africae, is the most common tick-borne rickettsiosis in sub-Saharan Africa. Mediterranean spotted fever due to Rickettsia conorii also occurs in the region but is more prevalent in Mediterranean countries. Using microimmunofluorescence, we compared the development of immunoglobulin G (IgG) and IgM titers in 48 patients with African tick-bite fever and 48 patients with Mediterranean spotted fever. Doxycycline treatment within 7 days from the onset of disease significantly prevented the development of antibodies to R. africae. In patients with African tick-bite fever, the median times to seroconversion with IgG and IgM were 28 and 25 days, respectively, after the onset of symptoms. These were significantly longer by a median of 6 days for IgG and 9 days for IgM than the times for seroconversion in patients with Mediterranean spotted fever (P < 10(-2)). We recommend that sera collected 4 weeks after the onset of signs of patients with suspected African tick-bite fever should be used for the definitive serological diagnosis of R. africae infections.

Anti-Bacterial Agents↗
PubMed2002

Value of microimmunofluorescence for diagnosis and follow-up of Bartonella endocarditis.

Bartonella endocarditis is a disease of emerging importance that causes serious complications and high rates of mortality. Due to the fastidious nature of Bartonella species and their high degrees of antibiotic susceptibility, cultures of clinical samples most often remain sterile and valvular biopsy specimens, the best specimens for PCR amplification, are seldom available. Therefore, serology appears to be the easiest diagnostic tool. In order to determine the best cutoff value for serology and its predictive values for the detection of Bartonella endocarditis, we studied 48 patients with culture- and/or PCR-confirmed Bartonella endocarditis. We also applied these serological criteria to 156 patients with blood culture-negative endocarditis. Furthermore, we compared the kinetics of the antibody responses to Bartonella spp. in order to estimate the value of serology for prediction of the occurrence of relapses. A titer of > or = 1:800 for immunoglobulin G antibodies to either Bartonella henselae or B. quintana has a positive predictive value of 0.810 for the detection of chronic Bartonella infections in the general population and a value of 0.955 for the detection of Bartonella infections among patients with endocarditis. When this cutoff was applied to 156 patients with blood culture-negative endocarditis, we were able to diagnose Bartonella infections in an additional 45 patients with definite endocarditis for whom a positive Bartonella serology was the only evidence of infection. On follow-up, the kinetics of the decrease in antibody titers were significantly delayed in two patients with relapses. In conclusion, we recommend the determination of antibodies to both B. quintana and B. henselae and the use of a cutoff value of 1:800 for the diagnosis of Bartonella endocarditis. We propose that this criterion, which may also help with the detection of late relapses, be included as a major criterion in the Duke criteria for the diagnosis of infective endocarditis.

Antibodies, Bacterial↗
PubMed2002

Determination of the nucleotide sequences of heat shock operon groESL and the citrate synthase gene (gltA) of Anaplasma (Ehrlichia) platys for phylogenetic and diagnostic studies.

The 1,670-bp nucleotide sequence of the heat shock operon groESL and the 1,236-bp sequence of the citrate synthase gene (gltA) of Anaplasma (Ehrlichia) platys were determined. The topology of the groEL- and gltA-based phylogenetic tree was similar to that derived from 16S rRNA gene analyses with distances. Both groESL- and gltA-based PCRs specific to A. platys were also developed based upon the alignment data.

Anaplasma↗
PubMed2002

Survival of Tropheryma whipplei, the agent of Whipple's disease, requires phagosome acidification.

Tropheryma whipplei was established as the agent of Whipple's disease in 2000, but the mechanisms by which it survives within host cells are still unknown. We show here that T. whipplei survives within HeLa cells by controlling the biogenesis of its phagosome. Indeed, T. whipplei colocalized with lysosome-associated membrane protein 1, a membrane marker of late endosomal and lysosomal compartments, but not with cathepsin D, a lysosomal hydrolase. This defect in phagosome maturation is specific to live organisms, since heat-killed bacilli colocalized with cathepsin D. In addition, T. whipplei survived within HeLa cells by adapting to acidic pH. The vacuoles containing T. whipplei were acidic (pH 4.7 +/- 0.3) and acquired vacuolar ATPase, responsible for the acidic pH of late phagosomes. The treatment of HeLa cells with pH-neutralizing reagents, such as ammonium chloride, N-ethylmaleimide, bafilomycin A1, and chloroquine, increased the intravacuolar pH and promoted the killing of T. whipplei. The ability of T. whipplei to survive in an acidic environment and to interfere with phagosome-lysosome fusion is likely critical for its prolonged persistence in host cells during the course of Whipple's disease. Our results suggest that manipulating the intravacuolar pH may provide a new approach for the treatment of Whipple's disease.

Acids↗
PubMed2002

Identification of Bartonella bacilliformis genotypes and their relevance to epidemiological investigations of human bartonellosis.

Genotypic diversity among 26 isolates of Bartonella bacilliformis obtained from different areas of Peru, and at different times, was assessed by comparison of DNA sequences derived from 16S-23S ribosomal DNA intergenic spacer regions (ISR) and a citrate synthase gene (gltA) fragment and by amplified fragment length polymorphism (AFLP) analysis. gltA comparison divided the isolates into two groups, whereas ISR comparison revealed six sequences. AFLP analysis using a selective primer delineated five profiles that correlated well with those obtained by sequence comparison. Combination of all three data sets divided the isolates into six genotypes. One of these genotypes was common to isolates collected from a large area in western Peru that corresponded to the region of endemicity for bartonellosis; however, isolates belonging to two other genotypes were also found within this region. Two of these genotypes were found in isolates isolated more than 35 years apart. The remaining three genotypes were each specifically associated with three outbreaks of bartonellosis that have recently occurred in areas where the disease had not previously been recognized. Demonstration of the unique nature of these isolates indicates that the outbreaks with which they were associated did not result from the introduction of disease by individuals who acquired their infection in the recognized region of endemicity. The sources of these outbreaks remain unknown. A consensus approach to bacterial typing using comparative sequence analysis of multiple genetic loci and the pan-genomic sampling of AFLP appears to offer a well-supported assessment of B. bacilliformis diversity, and the genotypic differences identified appear to have epidemiological significance.

Bacterial Proteins↗
PubMed2002

Improved culture from lymph nodes of patients with cat scratch disease and genotypic characterization of Bartonella henselae isolates in Australia.

Over a 4-year period we detected Bartonella henselae isolates in 104 of 297 specimens (35.1%) from Australian patients clinically suspected of having cat scratch disease by amplification of a fragment of the htrA gene. We isolated 17 B. henselae strains (20.5%) from the 83 PCR-positive human specimens available for culture. Our culture method was based on prolonged incubation in a moist atmosphere of blood agar to which hemin was added. We obtained more B. henselae isolates than the number of all other isolates from lymph nodes reported in the literature. In order to identify and study the genetic variation of Australian B. henselae isolates, we determined the sequence of a fragment of the pap31 gene from our 17 human isolates and also from 8 Australian cat isolates. Thirteen of the human B. henselae isolates belonged to the Houston genotype, variant Houston-1 (76.5%), and four belonged to the Marseille genotype, variant CAL-1 (23.5%). In contrast, seven cat isolates were classified as B. henselae Marseille, variant CAL-1 (87.5%), and one was classified as B. henselae Houston, variant Houston-1 (12.5%). Our study describes an efficient culture method for the diagnosis of cat scratch disease and contributes to the description of the genotypic distribution of B. henselae in Australia.

Australia↗
PubMed2002

Genetic classification and differentiation of Bartonella species based on comparison of partial ftsZ gene sequences.

Currently, 19 species are recognized in the genus Bartonella, 7 of which are involved in an increasing variety of human diseases. Development of molecular tools for detection, identification, and subtyping of strains and isolates has promoted research on Bartonella spp. We amplified and sequenced the portion of the ftsZ gene encoding the N-terminal region of the cell division protein for 13 Bartonella species: Bartonella alsatica, B. birtlesii, B. doshiae, B. elizabethae, B. grahami, B. koehlerae, B. schoenbuchensis, B. taylorii, B. tribocorum, Bartonella vinsonii subsp. arupensis, Bartonella vinsonii subsp. berkhoffii, Bartonella vinsonii subsp. vinsonii, and B. bovis Bermond et al.("B. weissii"). Phylogenetically derived trees revealed four statistically supported groups, indicating that sequencing of the ftsZ gene is a useful tool for identifying evolutionary relationships among Bartonella species. Furthermore, we amplified and sequenced the portion of the ftsZ gene encoding the C-terminal region of the protein for 4 B. bacilliformis isolates, 14 B. clarridgeiae isolates, 14 B. quintana isolates, and 30 B. henselae isolates that were obtained from different geographic regions, hosts, and clinical specimens. B. clarridgeiae and B. quintana sequences were highly conserved, while those of the four B. bacilliformis isolates differed from the type strain at 5 positions. Among B. henselae strains isolated from cats and patients, only two genotypes were detected: Houston and Marseille. Among 80 clinical samples we detected Bartonella spp. in 35 (43.75%) and found the assay to be comparable to that of a combined intergenic-spacer-region- and pap31-based PCR assay. Our results show the usefulness of the portion of the ftsZ gene encoding the C-terminal region for diagnosis of Bartonella infections. More samples should be tested to study its usefulness for epidemiological investigations.

Bacterial Proteins↗
PubMed2002

"Actinobaculum massiliae," a new species causing chronic urinary tract infection.

We report on a new Actinobaculum species, "Actinobaculum massiliae," isolated from the urine of an elderly woman with recurrent cystitis. Its phenotypic pattern was similar to those of both of the other Actinobaculum species described to date. On 16S rRNA sequencing, the Marseille isolate shared 95% homology with Actinobaculum suis, 92 to 93% homology with Actinobaculum schaalii, 91 to 92% homology with Arcanobacterium spp., and 87 to 90% homology with Actinomyces species. A bootstrap value of 99% supports the node separating the Actinobaculum sp. from its closest neighbor (A. suis). In conclusion, on the basis of phenotypic, genotypic, and phylogenetic assessments, we show that the Marseille isolate is a previously unrecognized organism within the Actinobaculum genus, and we propose placement of the organism in the taxon "Actinobaculum massiliae."

Actinomycetaceae↗
PubMed2002

Genomic variation of Bartonella henselae strains detected in lymph nodes of patients with cat scratch disease.

Bartonella henselae is the primary agent of cat scratch disease (CSD). In order to study the genetic variation of B. henselae and the correlation of the various genotypes with epidemiological and clinical findings, two seminested, groEL- and pap31-based PCR assays were carried out with specimens from 273 patients. Amplicons were sequenced to determine the genotype of the causative Bartonella species. Compared to our reference intergenic spacer region-based PCR, the groEL- and pap31-based assays were 1.7 and 1.9 times more sensitive, respectively. All 107 positive patients were infected with B. henselae; neither Bartonella clarridgeiae nor other species were detected. Based on the groEL and pap31 sequences, B. henselae amplicons were classified into two genogroups, Marseille and Houston-1, and into four variants, Marseille, CAL-1, Houston-1, and a new variant, ZF-1. Patients infected with either one or the other genogroup did not exhibit different epidemiological or clinical characteristics. Our study highlights the genotypic heterogeneity of B. henselae in patients with CSD.

Adolescent↗
PubMed2002

rpoB gene sequence-based identification of Staphylococcus species.

The complete sequence of rpoB, the gene encoding the beta subunit of RNA polymerase was determined for Staphylococcus saccharolyticus, Staphylococcus lugdunensis, S taphylococcus caprae, and Staphylococcus intermedius and partial sequences were obtained for an additional 27 Staphylococcus species. The complete rpoB sequences varied in length from 3,452 to 3,845 bp and had a 36.8 to 39.2% GC content. The partial sequences had 71.6 to 93.6% interspecies homology and exhibited a 0.08 to 0.8% intraspecific divergence. With a few exceptions, the phylogenetic relationships inferred from the partial rpoB sequences were in agreement with those previously derived from DNA-DNA hybridization studies and analyses of 16S ribosomal DNA gene sequences and partial HSP60 gene sequences. The staphylococcal rpoB sequence database we established enabled us to develop a molecular method for identifying Staphylococcus isolates by PCR followed by direct sequencing of the 751-bp amplicon. In blind tests, this method correctly identified 10 Staphylococcus isolates, and no positive results were obtained with 10 non-Staphylococcus gram-positive and gram-negative bacterial isolates. We propose partial sequencing of the rpoB gene as a new tool for the accurate identification of Staphylococcus isolates.

Bacterial Typing Techniques↗
PubMed2002

Genotypic characteristics of two serotypes of Bartonella henselae.

The study of 16S rRNA gene sequences of all isolates of Bartonella henselae obtained in our laboratory and others from human patients or cats has revealed two genotypes according to the sequence of the 16S rRNA gene. Two isolates of these genotypes have previously been related to two different serotypes, and lack of cross-protection of the two serotypes has been demonstrated in cats. We investigated the grouping of eight strains of B. henselae on the basis of 16S ribosomal DNA, 35-kDa protein, Pap 31 protein, and internal transcribed spacer (ITS) gene sequencing; sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles; and monoclonal antibody reactivity studies. Houston-1, 90-615, and SA2 strains showed the same patterns in SDS-PAGE, but they differed from the patterns of B. henselae isolates URBHLLY8, URBHLIE9, Cat6, Fizz, and CAL-1. Nine monoclonal antibodies derived from BALB/c mice immunized with B. henselae Houston-1 strain reacted only with strains Houston-1, 90-615, and SA2, and not with any other Bartonella strains. The two serogroups corresponded with two genotypes based on differences in the sequences of the genes encoding 16S rRNA, 35-kDa protein, and Pap 31 protein. Sequences of ITS genes were highly divergent among strains, as each had a unique sequence and the subdivision was not supported by DNA-DNA relatedness study. Study of 22 additional strains of B. henselae isolated from French bacteremic cats demonstrated that they all belong to one or the other of the proposed serotype or genotype.

Angiomatosis, Bacillary↗
PubMed2002

Genetic identification of rickettsiae isolated from ticks in Japan.

Following the description in Japan of Japanese spotted fever, caused by Rickettsia japonica, a search for the vector of this disease led to the isolation of several rickettsiae from various tick species. Sixty-three rickettsial isolates were obtained from six different tick species, and six type strains were described by PCR and monoclonal antibody testing. We identified these six strains by amplification and sequencing of the genes encoding 16S rRNA and citrate synthase. We confirmed that the isolates from Dermacentor taiwanensis and Haemaphysalis flava ticks were R. japonica isolates. In Ixodes ovatus, Ixodes persulcatus, and Ixodes monospinosus, we identified a Rickettsia identical or closely related to Rickettsia helvetica, a species that is pathogenic for humans and that to date has only been found in Europe. Finally, we identified a new genotype of unknown pathogenicity, genotype AT, that was isolated from Amblyomma testudinarium ticks and that is closely related to a Slovakian genotype obtained from Ixodes ricinus ticks.

Animals↗
PubMed2002

Detection of Rickettsia prowazekii in body lice and their feces by using monoclonal antibodies.

In order to identify Rickettsia prowazekii in lice, we developed a panel of 29 representative monoclonal antibodies selected from 187 positive hybridomas made by fusing splenocytes of immunized mice with SP2/0-Ag14 myeloma cells. Immunoblotting revealed that 15 monoclonal antibodies reacted with the lipopolysaccharide-like (LPS-L) antigen and 14 reacted with the epitopes of a 120-kDa protein. Only typhus group rickettsiae reacted with the monoclonal antibodies against LPS-L. R. felis, a recently identified rickettsial species, did not react with these monoclonal antibodies, confirming that it is not antigenically related to the typhus group. Monoclonal antibodies against the 120-kDa protein were highly specific for R. prowazekii. We successfully applied a selected monoclonal antibody against the 120-kDa protein to detect by immunofluorescence assay R. prowazekii in smears from 56 wild and laboratory lice, as well as in 10 samples of louse feces infected or not infected with the organism. We have developed a simple, practical, and specific diagnostic assay for clinical specimens and large-scale epidemiological surveys with a sensitivity of 91%. These monoclonal antibodies could be added to the rickettsial diagnostic panel and be used to differentiate R. prowazekii from other rickettsial species.

Animals↗
PubMed2002

Diagnosis of Wihipple disease by immunohistochemical analysis: a sensitive and specific method for the detection of Tropheryma whipplei (the Whipple bacillus) in paraffin-embedded tissue.

Whipple disease is a rare infection characterized clinically by diarrhea, fever, weight loss, arthralgia, malabsorption, and other systemic manifestations. The etiologic agent, Tropheryma whipplei, has been cultured only rarely. By using a polyclonal rabbit antibody produced against a cultured strain of T whipplei, tissue sections from 18 patients with Whipple disease were studied. Specimens from patients with histologic mimics and other infections served as control specimens. Immunostaining was identified in all 18 patients. Granular immunostaining was observed similar to that in periodic acid-Schiff (PAS) stains. In 2 patients, immunostaining was identified in specimens negative by H&E and PAS stains. In 4 patients studied before and after antibiotic therapy, immunostaining was retained but diminished in intensity and quantity. Immunostaining was not identified in any control specimen. Immunohistochemical analysis is a sensitive and specific method for the diagnosis of Whipple disease in paraffin-embedded tissue and may provide new opportunities to investigate the pathogenesis of the infection.

Actinobacteria↗
PubMed2002

Traditional and molecular techniques for the study of emerging bacterial diseases: one laboratory's perspective.

Identification of emerging bacterial pathogens generally results from a chain of events involving microscopy, serology, molecular tools, and culture. Because of the spectacular molecular techniques developed in the last decades, some authors think that these techniques will shortly supplant culture. The key steps that led to the discovery of emerging bacteria have been reviewed to determine the real contribution of each technique. Historically, microscopy has played a major role. Serology provided indirect evidence for causality. Isolation and culture were crucial, as all emerging bacteria have been grown on artificial media or cell lines or at least propagated in animals. With the use of broad-range polymerase chain reaction, some bacteria have been identified or detected in new clinical syndromes. Culture has irreplaceable advantages for studying emerging bacterial diseases, as it allows antigenic studies, antibiotic susceptibility testing, experimental models, and genetic studies to be carried out, and remains the ultimate goal of pathogen identification.

Animals↗