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
Subject(s)
Tuberculosis, Pulmonary , Tuberculosis, Multidrug-Resistant , Polymerase Chain ReactionABSTRACT
Spoligotyping is a method based on 36bp Direct Repeat [DR] chromosomal loci polymorphism which is connected to one or two 35-41 bp spacer sequences. There are 94 different intra DR spacer sequences which are identified so far and only 43 of them are used as usual. Mycobacterium tuberculosis complex strains can be identified based on lacking or having these sequences. Spoligotyping test was carried out on 238 TB smear positive patients. Primary separation of mycobacterium strains was done through Petrof 4% method and Lowenstein Jensen [LJ] media. Biochemical tests such as Niacin test/Catalase activity/Nitrate reduction were done in order to identify the strains. Drug sensitivity to INH [0.2Mg/ml]/ RIF [40Mg/ml]/ STM [10Mg/ml] and ETBl [2Mg/ml] identified by proportional method and according to that, the strains were divided into three groups: sensitive, multi drug resistance [MDR] and non MDR. Then DNA was extracted by CTAB method from the positive colonies. Sequences were amplified by PCR and after denaturizing, hybridization with Streptavidine peroxidase enzyme was performed by Line reverse blot method. Radiography was done after adding the Luminoscense and membrane onto the X-ray films. Serotypes were divided into 9 groups [Beijing/ CAS1/ Haarlem / U/ T2/ T1/ EAI3/ EAI2 and CAS2]. Most of the strains were from Haarlem [27%] and CAS1 [25%] groups. Two strains were also identified in this method that belonged to Mycobacterium bovis. Spoligotyping method is an easy, rapid and sensitive test in order to identify Mycobacterium tuberculosis complex strains