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Use of a New High Resolution Melting Method for Genotyping Pathogenic Leptospira spp
Florence Naze1, Amélie Desvars1, Mathieu Picardeau2
1Department of Microbiology, CHU de La Reunion, Saint-Pierre, La Réunion, France.
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.
Methods And Findings:
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.
Insights
A new genotyping method accurately identifies Leptospira species and subspecies. This technique, applied to Reunion Island samples, identified a unique genotype linked to major human leptospirosis cases.
Area of Science:
- Microbiology
- Infectious Diseases
- Molecular Diagnostics
Background:
- Leptospirosis is a global zoonotic disease prevalent in tropical regions.
- Leptospira interrogans is the main cause of human infections, with other species also implicated.
Purpose of the Study:
- To develop a rapid and accurate method for Leptospira species and subspecies identification.
- To apply this method to identify the primary cause of leptospirosis on Reunion Island.
Main Methods:
- Utilized melting curve analysis with specific primer pairs for species classification.
- Employed unsupervised high-resolution melting (HRM) combined with a statistical approach for subspecies typing.
- Applied variable-number tandem-repeat (VNTR) analysis for high-resolution genotyping.
Main Results:
- Accurate species classification of Leptospira reference strains was achieved.
- High-resolution genotyping at the serovar level demonstrated high discriminatory power (Hunter-Gaston index 0.984).
- Analysis of Reunion Island blood samples identified a unique genotype matching L. interrogans serovars Copenhageni and Icterohaemorrhagiae.
Conclusions:
- A simple, rapid, and robust genotyping method for Leptospira was established.
- The method allows for identification at both species and subspecies levels.
- Direct genotyping from biological samples without prior culturing is feasible.
