Molecular Detection of Isoniazid Gene among Patients with Multidrug Resistant Tuberculosis in Khartoum State

Abstract

The present study aimed to the detection of Isoniazid resistant gene (KatG) among MDR–tuberculosis by using polymerase chain reaction (PCR) techniques. Forty six isolates were obtained from suspected pulmonary tuberculosis patients attending the national tuberculosis reference laboratory. These isolates were subcultured on Lowenstein Jensen (LJ) medium and incubated at 37°C for 3 weeks and they were tested for the presence of acid fast bacilli using ZN-stain. All isolates showed obvious growth on LJ media with similar features of Mycobacterium tuberculosis complex (MTC) and gave positive ZN-stain. Further biochemical tests were performed, these tests include sensitivity to Para – Nitrobenzoic Acid (PNB), sensitivity to Thiophene – 2 – Carboxylic Acid Hydrazide (TCH), nitrate reduction test and catalase test, the results of these tests confirmed the presence of MTC. Further confirmations were conducted for all isolates by amplification of IS6110 insertion sequence by using PCR technique. The isolates showed bands equal to123bp which is typical to target sequence IS6110. All MTC isolates were subjected to drug susceptibility tests by conventional proportion method. The results showed that 20 (43.5%) were sensitive to the first line drugs (Isoniazid, Rifampicin, Streptomycin, Ethambutol), 20 (43.5%) were multidrug resistant (Isoniazid and Rifampicin), 3 (6.5%) were mono resistant to Streptomycin and 3 (6.5%) were found to have triple resistant to Isoniazid, Ethambutol and Streptomycin. Twenty six isolates that showed resistant pattern to different empirical antibiotics were subjected to PCR to targeting the KatG gene which is responsible for Isoniazid resistant. The obtained results showed that 23 (88.5%) isolates were positive for KatG gene, where as 3 (11.5%) isolates were negative for this gene that confirmed the results of the conventional method. From these results we concluded that the isolation of KatG gene can be considered as a good marker for the detection of multi drug resistant tuberculosis among suspected individuals.