Molecular epidemiology of indigenous isolates of Mycobacterium tuberculosis

Aim and objectives: To identify M. tuberculosis strain prevalent in Bhopal region using microbiological, immunological and molecular techniques. Rapid and accurate detection, identification and susceptibility testing of mycobacteria is important in Indian scenario due to increase in incidence of tuberculosis, resistance to antituberculous drugs and increase in potentially pathogenic species of mycobacterium. It is important to identify false positive reporting from clinical infections. To use Amplified ribosomal DNA restriction analysis (ARDRA) for identification of Mycobacterium species with different restriction enzymes and compare obtained profiles with a library of ARDRA profiles of different species. Microbiological and immunological studies are also important to identify the TB strains. Methodology: Sputum samples were collected from Peoples Hospital, Bhanpur, Bhopal after consent. The sputum was examined: MICROBIOLOGICALLY: AFB and culture on LJ media, IMMUNOLOGICALLY: Immuno-Dot Blot assay to detect serum Anti-Ag 38 and 65 KDa using secondary conjugate and secondary gold conjugate antibody specific for M. tuberculosis. MOLECULARLY: Genomic DNA Isolation, purification, Agarose Gel electrophoresis GENE AMPLIFICATION : Rep-PCR using primer BOX, Gm3f and Gm4r, ARDRA using restriction digestion with Alu I, Msp I and Taq I. Comparative analysis done using NTSYSpc version 2.02i, UPGMA method for phylogenetic relatedness among Mycobacterium species. Results: 138 suspected TB sputum samples microbiologically were all AFB positive. Culturing showed that 78 samples were of M. tuberculosis (MTB) and remaining of other mycobacterium species. Immuno-Dot Blot assay using secondary conjugate and gold conjugate showed 59 samples as MTB affected with elevated level 80-85μg/ml of 38/65 KDa antigen. M. intracellulare affected sample showed 73 μg/ml and healthy samples were below 50μg/ml. Genomic DNA was isolated from 117 samples. Rep-PCR showed banding for 124 samples with yield of 193 scorable bands ranging from 700bp - 300bp. 53 samples showed MTB, 39 MTB sub-strains and 34 belonged to other Mycobacterium species. 14 samples showed no banding pattern. In ARDRA the restriction patterns with the enzymes Alu I, Msp I and Taq I for the species presented banding pattern at 500, 300 and 100bp. 16S rRNA sequencing gave 99.8% similarity to MTB. It revealed mutation shifts of 500 and 300bp band replaced by a single fragment of 800bp. 16S rDNA amplification showed merged band of 1500bp for MTB. The AFB positive samples were also identified as non-tuberculous organism with ARDRA. Comparative analysis resulted in the formation of two clusters.