Use of rPOB Gene in the Diagnosis of Multi-Drug Resistant Tuberculosis in Sudan

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 and Gazeera States by using conventional methods also to identify rifampicin resistance M. tuberculosis by amplifying rpoB gene, using polymerase chain reaction (PCR). Moreover, it aimed to detect M. tuberculosis from direct sputum by PCR. Out of 228 of sputum samples, 128 and 100 were collected from suspected tuberculosis patients in Khartoum and Gezeera states respectively. Smears were made stained directly using Ziehl-Neelsen (ZN) stain. The results showed that, among Khartuom state suspected patients, 36 (21.7%) were positive for acid fast bacilli (AFB) while 92 (78.3) were negative, and among Gazeera state suspected patients 23(23%) were positive while 77 (77%) were negative. All sputum samples from Khartuom were inoculated on Lowenstein Jensen (LJ) medium and incubated aerobically at 37°C,the isolates showed obvious growth in 46 (36%) whereas 82 (64%) showed no growth, while samples from Gazeera were suspected to direct to PCR. Selected biochemical tests were performed to all Mycobacterium tuberculosis complex (MTC) isolates of Khartuom state, 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 123bp bands for IS6110 gene, Drug sensitivity tests were performed to all isolates, the results showed that 26(20.3%) 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 twenty six 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 20.3% of the mycobacterium tuberculosis isolates, of them 86.7% had resistance to rpoB gene. Regarding Gazeera state, out of 74 isolates the resistant strains were 25 (33%) only 19 (19%) gave band typical in size to the target gene rpoB (193bp) as indicated by standard DNA ladder for the present of rifampicin resistant gene, of them 76% had resistance to rpoB gene. 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 a valuable, rapid and sensitive technique which can replace conventional method, and PCR is also useful when taken directly from sputum. The study recommended that, PCR assay could be introduced as a diagnostic tool for tuberculosis, and recommended to support the development of new tools and enable their timely and effective use.