[ rapid identification of atypical mycobacterium in pulmonary tuberculosis [PTB] patients: evaluation of QUB3232 locus using the VNTR method]

ABSTRACT

Identification of atypical mycobacterium [Non tuberculosis Mycobacterium; NTM] is important because of the worldwide propagation of these organisms. Recently, molecular studies have identified the specific loci for mycobacterium species by DNA - finger printing methods, but these methods are time-consuming and expensive. In this study, in addition to hsp65 PCR-RFLP method, QUB3232 locus was evaluated for differentiation of atypical mycobacterium from mycobacterium tuberculosis complex. This study was performed on 371 pulmonary and non pulmonary specimens separated from patients with the symptoms of pulmonary tuberculosis [PTB]. After the isolation and culturing of mycobacterium strains using the Lowenstein Jensen media, biochemical tests including production of Niacin, Catalase activity, Nitrate reduction, pigment production and growth rate were performed. Drug susceptibility testing was performed by proportional method. DNA extraction was performed by phenol-chloroform method. hsp65 gene was amplified by PCR. Subsequently the amplicons were digested with three restriction enzymes namely AvaII, HphI and HpaII and electrophoresed on 3% agarose gel. QUB3232 locus was also evaluated for differentiation of atypical mycobacterium and mycobacterium tuberculosis complex. Out of 371 isolates, 32 [8.6%] were multi-drug resistant TB [MDR-TB], 184 [49.5%] were susceptible and 155 [42.5%] were non MDR [combined resistance] that 15% of MDR cases and 25% of non MDR cases were non tuberculosis mycobacterium. Out of 31 slow growing isolates, 58% were M. simiae and 19% were M. kansasii. The sensitivity of QUB3232 locus for differentiation of the atypical mycobacterium from mycobacterium tuberculosis complex was 80%. From the total of 43 NTM samples, 12 [27.9%] were rapid growing and 72% were slow growing. QUB3232 locus has the high discriminative power for differentiation of atypical mycobacterium from the mycobacterium tuberculosis complex, therefore, it can be used as a substitute for PCR-RFLP method: