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Sainte-Foy-lès-Lyon, France

Compain F.,Microbiology | Compain F.,University of Paris Descartes | Compain F.,French Institute of Health and Medical Research | Bruneval P.,Microbiology | And 15 more authors.
New Microbes and New Infections | Year: 2015

Endocarditis due to Legionella spp. is uncommon but presumably underestimated given the prevalence of Legionellae in the environment. We report a first and unusual case of chronic native valve endocarditis due to L. anisa and advocate that the diagnosis of endocarditis be made collaboratively between the cardiologist, surgeon, microbiologist and pathologist. © 2015 The Authors. Source

Gomgnimbou M.K.,University Paris - Sud | Gomgnimbou M.K.,Center Muraz | Ginevra C.,University of Lyon | Ginevra C.,French Institute of Health and Medical Research | And 17 more authors.
Journal of Clinical Microbiology | Year: 2014

A 42-plex clustered regularly interspaced short palindromic repeat (CRISPR)-based typing technique (spoligotyping) was recently developed at the French National Reference Center for Legionella. It allows the subtyping of the Legionella pneumophila sequence type 1/Paris pulsotype. In this report, we present the transfer of the membrane-based spoligotyping technique to a microbead-based multiplexed format. This microbead-based high-throughput assay uses devices such as Luminex 200 or the recently launched Magpix system (Luminex Corp., Austin, TX). We designated this new technique LP-SPOL (for L. pneumophila spoligotyping). We used two sets of samples previously subtyped by the membrane-based spoligotyping method to set up and validate the transfer on the two microbead-based systems. The first set of isolates (n=56) represented the whole diversity of the CRISPR patterns known to date. These isolates were used for transfer setup (determination of spacer cutoffs for both devices). The second set of isolates (n=245) was used to validate the transfer to the two microbead-based systems. The results obtained by the Luminex 200 system were 100% concordant with those obtained by the Magpix system for the 2 sets of isolates. In total, 10 discrepant results were observed when comparing the membrane-based method to the microbead-based method. These discrepancies were further resolved by repeating either the membrane-based or the microbead-based assay. This new assay is expected to play an emerging role for surveillance of L. pneumophila, starting with one of the most frequent genotypes, the sequence type 1/Paris pulsotype. However, the generalization of this typing method to all L. pneumophila strains is not feasible, since not all L. pneumophila strains contain CRISPRs.Copyright © 2014, American Society for Microbiology. All Rights Reserved. Source

Den Boer J.W.,Regional Public Health Laboratory Kennemerland | Euser S.M.,Regional Public Health Laboratory Kennemerland | Nagelkerke N.J.,United Arab Emirates University | Schuren F.,Applied Scientific Research | And 2 more authors.
BMC Genomics | Year: 2013

Background: Legionella is a water and soil bacterium that can infect humans, causing a pneumonia known as Legionnaires' disease. The pneumonia is almost exclusively caused by the species L. pneumophila, of which serogroup 1 is responsible for 90% of patients. Within serogroup 1, large differences in prevalence in clinical isolates have been described. A recent study, using a Dutch Legionella strain collection, identified five virulence associated markers. In our study, we verify whether these five Dutch markers can predict the patient or environmental origin of a French Legionella strain collection. In addition, we identify new potential virulence markers and verify whether these can predict better. A total of 219 French patient isolates and environmental strains were compared using a mixed-genome micro-array. The micro-array data were analysed to identify predictive markers, using a Random Forest algorithm combined with a logistic regression model. The sequences of the identified markers were compared with eleven known Legionella genomes, using BlastN and BlastX; the functionality for each of the predictive markers was checked in the literature.Results: The five Dutch markers insufficiently predicted the patient or environmental origin of the French Legionella strains. Subsequent analyses identified four predictive markers for the French collection that were used for the logistic regression model. This model showed a negative predictive value of 91%. Three of the French markers differed from the Dutch markers, one showed considerable overlap and was found in one of the Legionella genomes (Lorraine strain). This marker encodes for a structural toxin protein RtxA, described for L. pneumophila as a factor involved in virulence and entry in both human cells and amoebae.Conclusions: The combination of a mixed-genome micro-array and statistical analysis using a Random Forest algorithm has identified virulence markers in a consistent way. The Lorraine strain and related Dutch and French Legionella strains contain a marker that encodes a RtxA protein which probably is involved in the increased prevalence in clinical isolates. The current set of predictive markers is insufficient to justify its use as a reliable test in the public health field in France. Our results suggest that genetic differences in Legionella strains exist between geographically distinct entities. It may be necessary to develop region-specific mixed-genome microarrays that are constantly adapted and updated. © 2013 Den Boer et al.; licensee BioMed Central Ltd. Source

Baume M.,Center National Of Reference Des Legionelles | Garrelly L.,Biocontrol | Facon J.P.,Bio Rad | Bouton S.,rue du Courtil Center daffaires 35170 | And 4 more authors.
Journal of Applied Microbiology | Year: 2013

Aims: The characterization and certification of a Legionella DNA quantitative reference material as a primary measurement standard for Legionella qPCR. Methods and Results: Twelve laboratories participated in a collaborative certification campaign. A candidate reference DNA material was analysed through PCR-based limiting dilution assays (LDAs). The validated data were used to statistically assign both a reference value and an associated uncertainty to the reference material. Conclusions: This LDA method allowed for the direct quantification of the amount of Legionella DNA per tube in genomic units (GU) and the determination of the associated uncertainties. This method could be used for the certification of all types of microbiological standards for qPCR. Significance and Impact of the Study: The use of this primary standard will improve the accuracy of Legionella qPCR measurements and the overall consistency of these measurements among different laboratories. The extensive use of this certified reference material (CRM) has been integrated in the French standard NF T90-471 (April 2010) and in the ISO Technical Specification 12 869 (Anon 2012 International Standardisation Organisation) for validating qPCR methods and ensuring the reliability of these methods. © 2013 The Society for Applied Microbiology. Source

Descours G.,University of Lyon | Descours G.,French Institute of Health and Medical Research | Descours G.,Center National Of Reference Des Legionelles | Suet A.,University of Lyon | And 18 more authors.
Journal of Clinical Microbiology | Year: 2012

We evaluated the contribution of amoebic coculture to the recovery of Legionella spp. from 379 respiratory samples. The sensitivity of axenic culture was 42.1%. The combination of axenic culture with amoebic coculture increased the Legionella isolation rate to 47.1%. Amoebic coculture was particularly efficient in isolating Legionella spp. from respiratory samples contaminated with oropharyngeal flora. Copyright © 2012, American Society for Microbiology. All Rights Reserved. Source

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