Molecular genetics of Mycobacterium tuberculosis resistant to aminoglycosides and cyclic peptide testing by MTBDRsl...

The GenoType MTBDRsl test rapidly detects resistance to ethambutol, fluoroquinolones, second-line aminoglycosides (amikacin [AMK] and kanamycin [KAN]), and cyclic peptides (capreomycin [CAP]) in Mycobacterium tuberculosis. According to data from Global Drug Resistance Surveillance Report (2007), Armenia is counted as a high-burden country for multidrug-resistant tuberculosis (MDR-TB). The estimated burden of MDR-TB in 2012 was 9.4 (7-12) and 43 (38-49) among retreatment TB cases. A total of 92 laboratory confirmed cases were reported to the World Health Organization (57 new and 35 previously treated) out of 511 cases tested for MDR-TB.

A set of 77 drug-resistant TB isolates during 2011 and 2012 period, being either acid-fast bacterium positive or negative but culture-positive resistant to isoniazid, rifampin, or both according to the GenoType MTBDR plus assay, were consecutively tested using GenoType MTBDRsl. rrs gene analysis and the results from GenoType MTBDRsl were compared with phenotypic drug resistance testing. The DNA preparation method was performed as recommended by the manufacturer (Genotype MTBDR plus version 1.0 and Genotype MTBDRsl version 2.0 Hain Lifescience Nehren, Germany).

Aminoglycosides are key drugs for the treatment of MDR-TB. A total of 77 drug-resistant TB and four extensively drug-resistant M. tuberculosis isolates from Armenian TB patients were analyzed to characterize mutations within rrs and to compare with phenotypic drug resistance testing. Simultaneously, the following were identified: 65 (84.41%) rrs wild type (WT), 1 (1.3%) rrs WT MUT1 and MUT2 (WT; A1401G and G1484T), 1 (1.3%) rrs WT1, MUT1 (A1401G), 9 (11.7%) rrs WT1, MUT1 (A1401G), and 1 (1.3%) rrs WT1, MUT1. Mutation at position 1401 in rrs leads to resistance to KAN (7/77=9%), AMK (9/77=11.68%), and CAP (5/77=6.49%). Eleven (14.28%) streptomycin-resistant strains had a rrs mutation.

Isolates with rrs structural gene mutations were cross-resistant to streptomycin, KAN, CAP, and AMK. Detection of the A1401G mutation appeared to be 100% specific for the detection of resistance to KAN and AMK. Being the first assessment, these data establish the presence of phenotypic drug-resistant and extensively drug-resistant strains using molecular profiling and are helpful in understanding aminoglycoside resistance on a molecular level.