Name of Student: Zirwatul Adilah Binti Aziz
Name of Supervisor: Prof. Yap Sook Fan
Name of Co-Supervisor: Ms. Leila Hillout
Title: Molecular characterisation of multi-drug resistant isolates of Mycobacterium tuberculosis in
Malaysia
Abstract
The aims of this study are (i) to analyse the prevalance and distributions of the most commonly
reported mutations associated with rifampicin(RIF) and isoniazid (INH) resistance, (ii) the
relationship between such phenotypes and the patterns of genetic mutations in the corresponding
resistance genes (rpoB (RIF), katG and inhA (INH), and (iii) to determine the genetic diversity of
MDR-TB strains in Malaysia.
Methods : In this study, local isolates of M. tuberculosis found to be RIF and INH resistant by
Absolute Concentration drug susceptibility testing method will be studied. The minimun inhibitory
concentration (MIC) values of the aforementioned anti-tuberculosis drugs will be measured using
Middlebrook 7H9 broth containing serial dilutions of RIF (0.06µg/ml to 64 µg/ml) and INH (0.05
µg/ml to 3.2 µg/ml) for each of MDR-TB isolates. Genetic mutations in the rpoB gene of these MDRTB isolates will be identified by DNA sequencing of the purified PCR products. For detection of
isoniazid resistance gene, three approaches will be used. Detection of mutation(s) in katG gene will
be carried out by analyses of a portion of the katG gene after PCR amplification and digestion with
the restriction enzyme MspI. The detection of inhA mutation(s) will involve amplification of this gene
followed by DNA sequencing of PCR products. Multiplex PCR followed by sizing of the fragments will
be used for detection katG gene and inhA mutations. The size of PCR fragments will be estimated
following electrophoresis in 2 % NuSieve agarose gel. For studying the molecular epidemiology, DNA
fingerprinting analysis will be carried out using two methods, Mycobacterial Interspersed Repetitive
Unit-Variable Number Tandem Repeat (MIRU-VNTR) and spoligotyping.
Expected Results :
(i)
Based on the results of the gene mutation analysis, it is possible that hitherto unknown
drug resistance-associated mutations may be discovered
(ii)
This in turn may lead to the alteration of existing testing protocols to provide for a more
rapid and comprehensive detection of drug resistance among MTB isolates in Malaysia
(iii)
Strain differentiation with Spoligotyping and MIRU-VNTR typing will increase
understanding of the epidemiology of TB and MDR-TB in Malaysia