Detection of Primary Drugresistance Mutations Mycobacterium Tuberculosis in Khartoum State

Abstract

In Sudan as in many other countries the emerging drug resistant tuberculosis is causing a major public health threat. In the present study 250 sputum samples randomly collected from new tuberculosis patients were collected before administration of drugs from patients of different age groups of both sexes. The patients were from Khartoum Teaching Chest Hospital and Hamed El-Nile Hospital. The samples were collected during the period July 99 to May 2000. All studied patients were clinically diagnosed as tuberculosis cases. The samples were collected to study the primary drug resistance mutation in M. tuberculosis complex. 201 out of the 250 samples (80.5%) were found to have acid fast bacilli (AFB) on microscopy. 147 out of the 201 AFB positive (73.1%) gave growth of M. tuberculosiscomplex on Löwenstein Jensen medium (LJ) and three (6.1%) out of 41 which were not (AFB). 17 of the isolates (8.5%) were rapid grower mycobacteria other than tuberculosis (MOTT). Seven (3.5 %) were Nocardiasp. which were fully identified using biochemical test and molecular methods such as sequencing. The species was discovered to be a new entityand given a new name (Nocardia africana)by the International Taxonomy for bacteria, and 14 (6.9%) of the samples shown were contaminated cultures and 16 (7.9%) of the samples did not show any growth. The polymerase chain reaction (PCR), amplification of IS6110and the Restriction Fragment Length polymorphism (RFLP)-fingerprinting using PVUII enzyme was used to confirm the above phenotypic identify of the mycobacteria, using these techniques, 150 isolates were found to be M. tuberculosis complex. DNA from all the 150 M. tuberculosisisolates was extracted and then amplified by PCR using specific primers (TR8, TR9) to amplify rpoBgene around codon 43. Mutation in this gene is known to confer resistance to rifampicin. Single Strand Conformation Polymorphism (SSCP) technique was used to detect the presence of mutations among the 150 M. tuberculosisstrains. Only four strains showed mobility shift due to rpoBmutant gene. This is indicative of primary drug resistance to rifampicin. The analysis of rpsL gene around codon 43 by PCR- RFLP to the 150 M. tuberculosisstrains revealed that only two isolates of M. tuberculosisdid not cut with the restriction enzyme MboII. This indicates that these two strains have mutation at the rpsLgene which is responsible for resistance to streptomycin. V The rate of primary mutation among the 150 M. tuberculosisstrains isolated from new tuberculosis (received no treatment) was thusfound to be 2.7 %, 1.33 % for rpoBand rpsLgenes, respectively. And the rate of both genes was found to be (0.7%)