title: Use of a New High Resolution Melting Method for Genotyping Pathogenic Leptospira spp.
abstract: BACKGROUND
  Leptospirosis is a worldwide zoonosis that is endemic in tropical areas, such as Reunion Island. The species Leptospira interrogans is the primary agent in human infections, but other pathogenic species, such as L. kirschner and L. borgpetersenii, are also associated with human leptospirosis.
  
  
  RESULTS
  In this study, a melting curve analysis of the products that were amplified with the primer pairs lfb1 F/R and G1/G2 facilitated an accurate species classification of Leptospira reference strains. Next, we combined an unsupervised high resolution melting (HRM) method with a new statistical approach using primers to amplify a two variable-number tandem-repeat (VNTR) for typing at the subspecies level. The HRM analysis, which was performed with ScreenClust Software, enabled the identification of genotypes at the serovar level with high resolution power (Hunter-Gaston index 0.984). This method was also applied to Leptospira DNA from blood samples that were obtained from Reunion Island after 1998. We were able to identify a unique genotype that is identical to that of the L. interrogans serovars Copenhageni and Icterohaemorrhagiae, suggesting that this genotype is the major cause of leptospirosis on Reunion Island.
  
  
  CONCLUSIONS
  Our simple, rapid, and robust genotyping method enables the identification of Leptospira strains at the species and subspecies levels and supports the direct genotyping of Leptospira in biological samples without requiring cultures.
authors.affiliation: Department of Microbiology, CHU de La Reunion, Saint-Pierre, La Réunion, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Naze
authors.affiliation: Department of Microbiology, CHU de La Reunion, Saint-Pierre, La Réunion, France.
authors.forename: Amélie
authors.initials: A
authors.lastname: Desvars
authors.affiliation: Unité de Biologie des Spirochètes, National Reference Center and WHO Collaborating Center for Leptospirosis, Institut Pasteur, Paris, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Picardeau
authors.affiliation: Unité de Biologie des Spirochètes, National Reference Center and WHO Collaborating Center for Leptospirosis, Institut Pasteur, Paris, France.
authors.forename: Pascale
authors.initials: P
authors.lastname: Bourhy
authors.affiliation: Department of Microbiology, CHU de La Reunion, Saint-Pierre, La Réunion, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Michault
mesh_terms: D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000097:blood / Q000235:genetics; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D007919:Leptospira / Q000235:genetics*; D018598:Minisatellite Repeats; D009691:Nucleic Acid Denaturation; D060888:Real-Time Polymerase Chain Reaction; D012984:Software; D013045:Species Specificity
chemical_list: D004269:DNA, Bacterial