Genotyping Mycobacterium tuberculosis isolates with few copies of IS6110: Value of additional genetic markers.

ABSTRACT

PURPOSE: IS6110 restriction fragment length polymorphism (RFLP) analysis is widely used for molecular epidemiological studies of tuberculosis. Role of spoligotyping and Fluorescent Amplified Fragment Length Polymorphism (FAFLP) was studied in low-copy number IS6110 strains of Mycobacterium tuberculosis complex (Mtbc). METHODS: The study isolates included 70 strains of Mtbc collected from different regions of India. IS6110 restriction fragment, spoligotyping and FAFLP were performed for genotypic analysis. RESULTS: A single copy of IS6110 was found in 30% of isolates with 90.5% of them harboring characteristic 1.5-Kb IS6110 restriction fragment.IS6110RFLP identified 51 different types, FAFLP 41 types, and spoligotyping 31 types. Combination of all three techniques identified 67 different types.IS6110 RFLP analysis was found sensitive for genotyping isolates with more than one copy of IS6110 (Hunter Gaston Discriminatory Index (HGDI-1) while, neither spoligotyping (HGI-0.89) nor FAFLP (HGDI-0.92) or their combinations were as good. The discriminatory power of spoligotyping (HGDI- 0.89) in isolates with a single copy of IS6110 was higher than IS6110-RFLP.Clustering was reduced to 67% using spoligotyping and to 38% with FAFLP. CONCLUSION: Combination of FAFLP and Spoligotyping may prove to be valuable in studying the epidemiology of M. tuberculosis strains harboring few copies of IS6110 element.