We describe a multiplex allele-specific (MAS)-PCR assay to detect simultaneously mutations in the first and third bases of the embB gene codon 306ATG. These mutations are known to confer ethambutol (EMB) resistance in the majority of clinical Mycobacterium tuberculosis isolates worldwide. The mutated bases are revealed depending on the presence or absence of the respective indicative fragments amplified from the embB306 wild-type allele. Initially optimized on purified DNA samples, the assay was tested on crude cell lysates and auramine-stained sputum slide DNA preparations with the same reproducibility and interpretability of the generated profiles in agarose gel electrophoresis. Since EMB resistance is generally linked to multiple-drug resistance (MDR), the MAS-PCR assay for EMB resistance detection can be used in clinical laboratory practice in areas with a high prevalence and a high transmission rate of MDR-EMB-resistant tuberculosis.is a first-line drug used for antitubercular therapy in combination with other drugs as recommended by the World Health Organization DOTS/DOTS-Plus regimens, as well as by the standard treatment protocol officially adopted by the Russian Ministry of Health. The EMB action on tubercle bacilli is bactericidal and is due to its interactions as an arabinose analogue with the target arabinosyl transferase (1, 9). As a result, the synthesis of sugars (arabinan and, consequently, arabinogalactan and lipoarabinomannan), necessary for cell wall construction, is prevented. Finally, the accumulation of mycolic acids results in cell death. The Mycobacterium tuberculosis emb operon encoding different arabinosyl transferases includes three contiguous genes, namely, embC, embA, and embB that exhibit 65% similarity to each other (9). Analysis of EMB r clinical isolates of M. tuberculosis identified several mutations in these genes conferring EMB resistance, mostly in embB, and up to 90% of them were found in codon 306ATG-Met (5, 8). Five different mutations were uncovered in this codon (ATG 3 GTG/CTG/ATA, ATC, and ATT), resulting in three different amino acid shifts (Met 3 Val, Leu, or Ile) (8). EmbB Met306 is located in a cytoplasmatic loop that forms an EMB resistance-determining region (9), and embB mutations undoubtedly mediate EMB resistance rather than act simply as surrogate markers of resistance (5). About 30% of EMB r strains had no embB mutations (5), and a recently undertaken extensive study suggested more genes are involved in EMB resistance in some strains (4). However, about one-fourth of EMB r strains still lacked any known mutation inferred to participate in EMB resistance, implying multiple molecular pathways to the EMB resistance phenotype, and some of them remain to be discovered.The prevalence of resistance to EMB, initial and acquired, is not generally high and varies in different countries from 1.3% in Canada (6) to 23.3% in Pakistan (3), with an overall average of 4 to 9% (for more information about initial resistance, see references 2, 5, 14, and 15), and is predominant ...