ABSTRACT
Multidrug-resistant tuberculosis [MDR-TB] is an emerging problem with high mortality rate where recently developed molecular techniques represent potential tools for its early detection. The aim of this study is to detect drug resisting mutants of Mycobacterium tuberculosis [M.TB] in 15 patients with active pulmonary tuberculosis, who were non responding to 1[st] line multi-drug therapy [refampicin [ RIF] and isoniazid [INH]]. This was performed using the Chain Termination method of manual Gene Sequencing for the following Mycobacterial genes: rpoB gene [involved in sensitivity to RIF], katG and inhA genes [involved in sensitivity to INH]. Missence point mutations in rpoB gene were found in 93.3 % [14/15], which involved codon 184 in 80% [12/15] with Histidine-Tyrosine substitution, and codon 174 in only 13 .3% [2/15] with Aspartate-Valine substitution. Missence mutations in katG gene were detected in all cases [100%], which involved codon 315 in 80% [12/15] with Serine- Threonine substitution and codon 444 in 13.3 % [2/15] with Valine - Alanine substitution, while only 6.7 %[1/15] involved codon 315 and 444 together. Inh-A gene revealed missence mutantion in 86.7% [13/15], where 60 % [9/15] involved codon 94 with Serine - Valine substitution, 20 %[3/15] involved codon 99 with proline - Arginine substitution and 6.7% [1/15] involved codon 69 with Glutamate - Alanine substitution. It could be concluded that, missence point mutations found in the examined genes could explain the resistance of 14 patients to both drugs and resistance of only one patient to INH alone. The missence point mutation was found at a common codon position among each gene, in addition to some other involved codons. It could be also concluded that, manual gene sequencing is a rapid, non expensive and accurate technique for early detection of MDR-TB, which helps early starting of proper treatment and inhibits spreading of such strains. Although it is difficult to be performed as a routine test, facilities should be available in order to perform it for at least some selected cases, as it does not need the expensive automated DNA sequencer