The usefulness of DR, PGRS and spoligotyping in the typing of Mycobacterium tuberculosis. Comparison with IS6110

Background: To evaluate 4 markers (IS6110, DR, PGRS and spoligotyping) to differentiate the strains of Mycobacterium tuberculosis isolated in our surroundings, most specially in those which contain a reduced number of IS6110 copies. In addition, to confirm the identity of the strains that share the same IS6110 restriction-hybridization pattern. Methods: We selected 37 strains from a previous study: 25 had a unique IS6110 pattern and 12 grouped in 3 clusters (8 strains with 11 bands, 2 with 17 bands and 2 which shared the same six-band pattern). The PGRS and DR-RFLPs were obtained by AluI restriction and synthetic oligonucleotides specific to these sequences. The polymorphism of the DR region spacers was analyzed by spoligotyping. For the amplification of the spacers we used the DRa and DRb primers. Detection was done hybridizing the PCR products on a membrane in which 43 specific spacers had been previously immobilized. Results: Twenty-three different PGRS patterns and 18 spoligotyping patterns were obtained from the 25 strains with unique IS6110 pattern. Eight patterns resulted from the 10 strains studied by DR. The 8 strains which shared an 11-band pattern, as well as the 2 strains which shared a 17-band pattern, resulted identical by the other markers. However, 2 strains which shared a 6-band pattern were different by both PGRS and DR or spoligotyping. Conclusions: 1) IS6110 resulted the most discriminative marker of all. 2) The clonality of clusters with a low number of bands has to be confirmed with alternative markers.