Identification of Multidrug-Resistant Mycobacterium tuberculosis; Comparison of The Standard Susceptibility and The DNA-­ Based Methods

Abstract

Tuberculosis (TB), though curable, still remains a major killer disease worldwide. One of the most • alarming trends concerning tuberculosis is the emergence of drug-resistant M tuberculosis strains, which has become a worldwide health care problem (Pablos-Mendez et al., 1998).

The •aim of this study was to compare the agar proportion and SSCP methods for the detection of multiple drug resistance (MDR) in Mtuberculosis. A second goal was to characterize the mutations in rpoB and katG genes associated with RIF and INH resistance by DNA sequencmg.

This study was conducted on 40 MTB isolates collected froni smear-positive pulmonary tuberculosis patients from lsmailia and Suez Chest Hospitals. Drug susceptibility testing was performed on the MTB isolates against 4 anti-tuberculosis drugs (INH, RIF, SM and EMB) in concentrations of 0.2, 40, 8, 2 1-1g/ml respectively by the L-J agar proportion method. The highest rate of resistance was that for SM (47.5%) while the lowest was for EMB (25%). The rate of MDR (resistance to both INH and RIF) was 35%.

SSCP detected 13 INH resistant isolates (32.5%) while it detected 12 (30%) RIF resistant ones out of 40 MTB isolates. When comparing the results of SSCP to those of the agar proportion method (the gold standard), we found that its sensitivity was 72.2% and 80% when testing for katG and rpoB mutations respectively, its specificity was I 00% and the 2 tests showed moderate to good agreement (Kappa= 0.74 and 0.83). I

As regards the type of mutations and the resulting amino acid'

substitutions associated with drug resistance, DNA

. sequencing

' revealed

that 13 of 14 INH-resistant isolates (92.8%) had mutations in katG; 1:2 had a S315T amino• acid substitution and the remaining one isolate had a • Dn9A substitution. Of the 13 , RIF-resistant isolates, shown to have mutations in rpoB by sequencing, 10 (77%) had a S 531L substitution and3 isolates (23%) had H526Y, H526L and Ss31 W substitutions. i