Rapid detection of ethambutol-resistant Mycobacterium tuberculosis strains by PCR-RFLP targeting embB codons 306 and 497 and iniA codon 501 mutations

Mutations at embB gene codons 306 and 497 and iniA gene codon 501 occur frequently in ethambutol (EMB)-resistant Mycobacterium tuberculosis strains worldwide. The identification of these mutations in resistant strains has been achieved by labor-intensive DNA sequencing or by tedious amplification protocols followed by restriction endonuclease digestion. In this report, we describe PCR-restriction fragment length polymorphism (RFLP)-based methods for determining substitutions at embB codons 306 and 497 and iniA codon 501 directly in BACTEC cultures of M. tuberculosis isolates. The wild-type and mutant alleles are revealed by easily interpretable and different RFLP patterns. The methods optimized initially on reference strains were tested directly on BACTEC cultures of 25 randomly selected clinical M. tuberculosis isolates, seven of which were determined to contain EMB-resistant strains by phenotypic drug susceptibility testing. The PCR-RFLP methods identified mutations in four of seven EMB-resistant strains with three isolates containing mutated embB codon 306 and one isolate containing mutated embB codon 497. The results of PCR-RFLP were confirmed by DNA sequencing. The worldwide prevalence figures for mutations at embB codons 306 and 497 and iniA codon 501 suggest that nearly half of EMB-resistant M. tuberculosis strains could be identified within one working day even in developing countries equipped with simple PCR technology instead of weeks required for phenotypic drug susceptibility testing. Further, since EMB resistance is also associated with multiple-drug resistance from some geographical locations, detection of EMB resistance may also lead to rapid identification of multidrug-resistant strains of M. tuberculosis.