Molecular Detection of Rifampicin –Resistance Gene in Mullti-drug Resistant Mycobacterium tuberculosis in Khartoum State

Abstract

The spread of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis has become a major public health concern since these bacteria often cause incurable disease, even when expensive second- and third-line drugs are available. This study aimed to identify M. tuberculosis among suspected tuberculous patients in Khartoum State by using conventional methods also to identify rifampicin resistance M. tuberculosis by amplifying (rpo B) gene, using polymerase chain reaction (PCR). 128 sputum samples were collected from suspected tuberculosis patients. Direct smears were performed by using ZN stains, the results showed that 36 (21.7%) were positive for AFB while 92 (78.3) were negative. All sputum samples were inoculated on Lowenstein Jensen medium and incubated aerobically at 37°C,the isolates showed obvious growth in 46 (36%) whereas 82 (64%) showed no growth. Selected biochemical tests were performed to all Mycobacterium tuberculosis complex (MTC) isolates, the results revealed that all isolates were sensitive to Para-nitro benzoic acid ( growth was inhibited by PNB), resistant to Thiophene - 2 - Carboxylic Acid Hydrazide (TCH), positive for nitrate reduction and were Catalase negative at 68°C. All the forty six isolates that showed typical growth of MTC on LJ medium were subjected to PCR to amplify (IS 6110) gene .The results indicate clearly that all isolates showed positive results for (IS 6110), 123bp. Drug sensitivity tests were performed to all isolates, the results showed that 26 (56.5%) as MDR-TB, 16 (34.8%) as sensitive to rifampicin, Isoniazid, Ethambutol and streptomycin, 2 (4.4%) as resistance to streptomycin, and 2(4.4%) as triple resistance to Isoniazid, Ethambutol and streptomycin. The thirty resistant isolates were subjected to PCR searching for rifampicin resistance gene (rpoB) with band equal to 193bp in size, the results showed existence of this band in (86.7%). Due to the low sensitivity of ZN technique and the long time required to conduct Drug susceptibility Test (DST) through conventional method, the results concluded the PCR is evaluable, rapid and sensitive technique which can replace conventional method.