Stain differentiation of South African clinical isolates of Mycobacterium tuberculosis by restriction and amplified fragment length polymorphisms

Abstract

DNA fingerprinting of Mycobacterium tuberculosis strain has been used in combination with conventional epidemiologic investigation, which has improved the understanding of tuberculosis transmission. Restriction Fragment Length Polymorphism (RFLP) based on IS6110 probe has become a standard method of fingerprinting of M tuberculosis. Since the technique is labour intensive and the discriminatory power of IS611 0 fingerprinting method for strains habouring only one to five copies is poor, other typing methods for typing M tuberculosis should be evaluated. In this regard, Amplified Fragment Length Polymorphism (AFLP) has the potential to overcome many of the RFLP problems. The first objective was to determine the suitability of the RFLP and AFLP techniques and to study the extent of transmission of tuberculosis in a referral hospital in South Africa. A total of 47 M tuberculosis isolates were differentiated using RFLP technique. The same samples were typed using the PCR- based AFLP technique and results were compared. The second objective was to determine the prevalence of isoniazid (INH) resistance and estimate the incidence of recent transmission of the disease in the Eastern-Cape (EC) and North-West province (NW) by using the best suited technique. RFLP grouped the 47 typed M. tuberculosis isolates into five families and four clusters. AFLP grouped the analyzed isolates (previously typed by RFLP) into two groups based on the banding patterns observed. As a result of the low degree of genotypic variation among the AFLP band pattern of M tuberculosis isolates, AFLP seemed less promising for individual strain differentiation of M tuberculosis. This technique can be used in future for differentiation of Mycobacterial species and The prevalence of INH resistance was found to be 6.7% in the EC and 8.4% in the NW province. The magnitude of recent transmission in the Eastern Cape studied by RFLP method, was found to be at 22% among the positive tuberculosis isolates identified. Transmission of TB in NW province was associated with reactivation rather than recent transmission due to lack of clustering of strains in that region.