Molecular Detection of Mycobacterium Tuberculosis Complex from Sputum of Clinically Suspected Tuberculosis Cases
Background: Tuberculosis (TB) is a communicable disease and it ranks second of all infectious agents due to co-infection with HIV . The causative agent of tuberculosis is a group of mycobacteria known as Mycobacterium tuberculosis complex. Mycobacterium tuberculosis complex consist of M. tuberculosis, M. bovis, M. africanum, M. microti, M. canetti. In PCR study, Most commonly sensitivity is higher in smear positive samples (95-100%) rather than smear negative specimens (46-63%). OBJECTIVE: To detection of Mycobacterium tuberculosis complex by Line Probe Assay. Methods: The study was done from non-interventional approaching study of 50 suspected patients of tuberculosis had visited the TB chest/ DOTS centre. Sputum sample collected early morning in a wide- mouth container from the patients having history of cough more than 2 weeks. Methodology used Z-N staining and for detection of MTB complex was done using MTBDR plus assay, multiplex PCR DNA strip assay (Hain Lifescience, Nehren, Gernamy) which is commercially available. M. tuberculosis secreted an important protein is MPT64, a 24-kDa protein. The major culture filtrate protein (24-kDa) is MPT64 encoded by the RD2 region genes and has been exposed to be an exact antigen that differentiates the M. tuberculosis complex from the mycobacteria other than tuberculosis (MOTT) Species. Results: In 50 samples, out of which 10 (20%) were smear positive & 40(80%) were smear negative. Out of 10 smear positive, 9 (90%) were MTB (Mycobacterium tuberculosis) & 01 (10%) was NTM (Non-tuberculous Mycobacteria) by PCR method. Out of 40 smear negative, 30 (75%) were positive by PCR method. Out of 30, 28(93%) were MTB & 02(7%) were NTM. Rests of the 10(25%) samples were found negative for M. tuberculosis complex. Conclusions: This study proved that PCR is a specific and sensitive method in comparison of sputum microscopy after staining with Z-N technique and it helpful the clinicians ability to diagnose and treat the patients on time. This will ensure early treat to patients and prevent further transmission of disease.
2. Ramachandran R and Parmasivan CN (2003). What is new in the diagnosis of tuberculosis? Part I: Techniques for diagnosis of tuberculosis. Indian J Tuberculosis 50: 133-141. Riska
3. Revised National TB Control Programme(RNTCP), Annual states report; central disease, 2016.
4. Dhurba Giri in Bacteriology, microbiology Ziehl- neelsen; principle, procedure and reporting and modification ; 2016
5. Scarparo C, Piccoli P, Rigon A, Ruggiero G, Scagnelli M, Piersimoni C. Comparison of enhanced Mycobacterium tuberculosis amplified direct test with COBAS AMPLICOR Mycobacterium tuberculosis assay for direct detection of Mycobacterium tuberculosis complex in respiratory and extrapulmonary samples. J Clin Microbiol. 2000;38(4):1559-
6. Mangan JA, Sole KM, Mitchison DA, Butcher PD. An effective method of RNA extraction from bacteria refractory to disruption, including mycobacteria. Nuc Acids Res. 1997;25:675-676.
7. Alli OAT, Mangan JA, Butcher PD, Spreadbury CL. Optimisation of RNA extraction in Mycobacterium tuberculosis for studying intracellular gene expression. Afr J Clin Exp Microbiol. 2009;10(2):64-79.
8. Oyebode A.T. Alli et al; Study on Direct molecular detection of Mycobacterium tuberculosis complex from clinical samples- an adjunct to cultural method of laboratory diagnosis of tuberculosis: North American Journal of Medical ; 2011:3 281-288
9. S Sankar et al; Comparative evaluation of two polymerase chain reaction targeting genomic regions to detect Mycobacterium tuberculosis in sputum; 2010:28(4)
10. Aroma Oberoi Aruna Aggarwal et al Study on comparison of the Conventional Diagnostic Techniques BACTEC, Culture and PCR test for the diagnosis of tuberculosis, w.w.w jkscience. Org, 2009:11 ,24-26
11. Chakravorty S, Tyagi JS. Novel multipurpose methodology for detection of mycobacteria in pulmonary and extrapulmonary samples by smear microscopy, culture, and PCR. J Clin Microbiol. 2005;43(6):2697-2702.
Copyright (c) 2017 International Archives of BioMedical and Clinical Research
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.