Frequency of mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis clinical isolates from Sudan

This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Multidrug-resistant (MDR) strains of Mycobacterium tuberculosis represent an obstacle in tuberculosis control and stress the need for rapid and reliable diagnostic tools for drug susceptibility testing in clinical isolates [1]. Mutations in the 81-bp region of the rpoB gene are responsible for resistance to rifampicin, an essential drug for the therapy of tuberculosis (TB) [2]. Mycobacteria laboratory facilities in Sudan exist only in Khartoum state whereas TB patients from other states are referred to Khartoum state for diagnosis and treatment. The MDR-TB prevalence in Sudan was found in 5% of new cases and 24% of previously treated patients [3]. Recent development of the Xpert MTB/RIF test [4] and of the line probe assay [5] which span 81-bp fragment of the RNA polymerase beta subunit (rpoB) gene have allowed for the rapid detection of resistance to rifampicin (RIF). Knowledge on the geographical distribution of the rpoB resistant alleles is essential for the development of diagnostic tools. Although sequence analysis of the rpoB gene has been shown to be effective for detecting RIF-resistance alleles in over 90% of RIF-resistant isolates from different geographical regions [6,7], there is only limited information from Sudan [8]. In order to find Sudan-specific mutations, that could potentially be used for the optimization of the current detection tools of RIF resistance alleles, we determined in this study the mutation profile occurring within an 81-bp fragment of the rpoB gene, in a collection of MDR clinical isolates of Mycobacterium tuberculosis isolated from Khartoum, Sudan. Forty-nine clinical isolates consisting of RIF-resistant M. tuberculosis were isolated from sputum of TB patients during the period between 2007 and 2009, into the Khartoum state, Sudan. The drug susceptibility testing was performed using standard proportional method and the drugs tested were rifampicin, isoniazid (INH), ethambutol (EMB), and streptomycin (SM) [9]. Genomic DNA was extracted using the boiling method. An 81-bp region of the rpoB gene was amplified by polymerase chain reaction (PCR) [10] and DNA sequencing was performed by Macrogen Inc. (Seoul, South Korea) on a ABI3730XL DNA sequencer (Applied Biosystems, Foster City, USA). Water was used as a negative control to replace DNA, to rule out carry-over contamination of the amplicon throughout the steps of the protocol. We report here the determination of the …