INVESTIGADORES
RUYBAL paula
congresos y reuniones científicas
Título:
Reduced MLST scheme optimized for direct application on Leptospira spp. human clinical samples
Autor/es:
VARNI VANINA; CHIANI YOSENA; NAGEL ARIEL; RUYBAL PAULA; VANASCO BIBIANA; CAIMI KARINA
Lugar:
Amsterdam
Reunión:
Encuentro; 2nd ELS meeting on Leptospirosis and other rodent borne haemorrhagic fever; 2015
Institución organizadora:
European Leptospirosis Society
Resumen:
Leptospirosis is an infectious disease caused by different species of the genus Leptospira and is a zoonosis worldwide distributed. The traditional classification of strains of Leptospira spp. is based on serological methods having high complexity in implementation and outcome. Currently, alternative molecular methods for typing isolates have arisen. Among them, multilocus sequence typing (MLST) allows genotyping pathogenic Leptospira being one of its main advantages the overall comparison of isolates through their allelic profile. Recently, our group conducted a reassessment of such methods by which an optimized scheme of 7 loci was described. Because obtaining isolates is extremely difficult, particularly for human samples, the aim of this study was to adapt this scheme for direct application to clinical samples, increasing the number of samples analyzed and enable better epidemiological surveillance of this zoonosis.Serum samples and DNA extracted from sera from patients with positive serology or positive LipL32-qPCR were used; the samples were characterized to species level by 16S-rRNA PCR. From our reassessed 7 loci scheme (R7L), the 3 more discriminating loci (MLST reduced) were select for direct amplification on clinical samples. The typing scheme proficiency was studied from sequences previously used in generating the complete scheme. Three loci were selected and its concatenated sequences were applied for phylogenetic analysis (Maximum Likelihood, MEGA6).Phylogenetic analysis with previous sequences confirmed that the reduced scheme matches the whole scheme in the clustering of isolates of the same species; it also allows to recognize sub-groupings of isolates within each specific cluster, validating its potential for typing.A total of 61 clinical samples were studied. Thirty-six could be amplified by 16S rRNA PCR. It was found that 94% belonged to the major pathogenic species: L. interrogans, L. kirschneri and L. borgpetersenii. Positive amplification was obtained for the MSLT reduced scheme in 4 of them. Additionally, 5 samples could be amplified with only 1 or 2 of the 3 loci. Positive samples for the three loci belong to the species L. interrogans. Phylogenetic analysis allowed to correlate this samples with isolates belonging to serogroups Canicola and Icterohaemorraghiae.The results obtained here constitute an important precedent for the molecular epidemiology of Leptospira sp., as the development of tools for typing directly on clinical samples will significantly increase the information on circulating genotypes, and will simplify the diagnosis and epidemiological control of this disease.