Abstract
Background: Early diagnosis of pulmonary tuberculosis is difficult using conventional diagnostic method. Acid-fast bacillus (AFB) culture and smear microscopy are alone inadequate to diagnose of human immunodeficiency virus (HIV) -associated Tuberculosis (TB). In both test low sensitivity is a major drawback. Advances in molecular techniques, which rapidly identify Mycobacterium DNA in sputum, may overcome these obstacles.
Objective: Molecular characterisation of HIV associated tuberculosis compared to conventional methods.
Study Design: A cross-sectional study design.
Setting: King George’s Medical University, Lucknow, Uttar Pradesh and Dolphin (PG) Institute of Biomedical and Natural Sciences, Dehradun, Uttarakhand,
Method: The present study showed the laboratory finding of 250 sero-positive HIV patients carried out of department of Microbiology, Medical University, Lucknow and Dolphin (PG) Institute of Biomedical and Natural Sciences, Dehradun, Uttarakhand. Their HIV status confirmed by Enzyme linked immune Sorbent Assay (ELISA) test in antiretroviral therapy (ART) centre Medical University, Lucknow. The study consisted with HIV-positive patient, clinically diagnosed for tuberculosis cases. All sputum samples subjected to AFB smear microscopy, culture and drug-susceptibility testing by 1% proportion method on Lowenstein-Jensen (LJ) medium and polymerase chain reaction (PCR).
Results:Among 250 HIV positive sputum samples 48 (19.2%) were smear positive 54 (21.6%) were culture positive and 58 (23.4%), PCR positive. PCR was found high sensitive and specific as a diagnostic tool compare to smear microscopy and culture.
Conclusion:The present study shows the major advances in molecular techniques, which rapidly identify Mycobacterium DNA in sputa with in comparison to other methods.
Key words: Mycobacterial culture, polymerase chain reaction, acid-fast bacilli, Lowenstein-Jensen medium.
References
1. Boehme CC, Saacks S, O'Brien RJ, (2013) “The changing landscape of diagnostic services for tuberculosis” Semin Respir Crit Care Med.34(1):17-31.
2. Saiki RK,Schraf S,Faloona F, et al.(1985) Enzymatic amplification of B globin genobw sequences and restriction site analysis for diagnosis of sickle cell anemia. Science; 230: 1350-1354.
3. Revised National Tuberculosis Control Programme. Training Module for Medical Practitioners, (2006). Available at: www.tbcindia.nic.in/pdfs/Financial%20Training%20 Module.pdf.
4. Revised National TB Control Programme Training Manual for Mycobacterium tuberculosis Culture & Drug susceptibility testing, 2009.
5. Revised National Tuberculosis Control Programme. DOTS plus Guidelines, 2010. Available at: www.health.bih.nic.in/ Docs/Guidelines-DOTS-Plus.pdf.
6. Roberts GD. Mycobacteria and Norcardia. In: J. A. Washington II, editor. Laboratory procedures in clinical microbiology. New York : Springer Verlag, 1981 p. 365-406.
7. Telenti A, Marchesi F, Balz M, Bally F, Bottger EC, Bodmer T.(1993) Rapid identification of Mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J Clin Microbiol; 31 : 175-8
8. Singh JP, Verma R, Chaudhuri (2006) Random amplified polymorphic DNA (RAPD) analysis of Mycobacterium tuberculosis strains in India P - J. Vet. Sci.7:181-187
9. Montenegro SH, Gilman RH, Sheen P, Cama R, Cavides L, Hopper T,(2003) Improved detection of M.tuberculosis in Peruvian children by use of heminested IS6110 PCR assay. Clin Infec Dis ,36:16-23.
10. Eisenach KD, Sifford MD, Cave MD, Bates JH, Crawford JT. (1991) Detection of Mycobacterium tuberculosis in sputum samples using a polymerase chain reaction. Am Rev Respir Dis , 144: 1160–1163.
11. Assis N.C.S., Lopes M.L., Cardoso N.C., Costa M.M., Souza C.O., Lima K.V.B. (2007) Diagnóstic of molecular tuberculose pulmonar. J. Bras. Patol. Med. Lab.43(1):1–7.
12. Flores L.L., Pai M., Colfors J.R., Riley L.W. et al (2005) In-house nucleic acid amplification tests for the detection of Mycobacterium tuberculosis in sputum species: meta-analysis and meta-regression. BMC. Microbiol. 2005;5:55.
13. Marchi A.M., Juttel I.D., Kawacubo E.M., Dalmarco E.M., Blatt S.L., Cordova C.M.M. Evaluation of methods for detection and identification of Mycobacteriumspecies in patients suspected of having pulmonary tuberculosis. Braz. J. Microbiol., 39:613–618.
14. Sarmiento O.L., Weigle K.A., Alexander J., Weber D.J., (2003) Assessment by meta-analyses of PCR for diagnosis of smear-negative pulmonary tuberculosis. J. Clin. Microbiol., 41(7):3233–3240.
15. Bechnoosh A, Lieberman JM, Duke MB, Stutman.(1997) Comparison of Quantitative polymerase chain reaction, therapy for pulmonary tuberculosis. Diag Microb Infect Dis , 29:73-79.
16. Prasad, S. and Singhal, M. and Negi, et al (2012)“ Targeted detection of 65 kDa heat shock protein gene in endometrial biopsies for reliable diagnosis of genital tuberculosis” European Journal of Obstetrics Gynecology and Reproductive Biology, 160(2): 215-218.
17. Boehme CC, Nicol MP, Nabeta P, Michael JS, Gotuzzo E, et al (2011) Feasibility, diagnostic accuracy and effectiveness of decentralised use of the Xpert MTB/RIF test for diagnosis of tuberculosis and multidrug resistance: a multicentre implementation study.Lancet 377: 1495-1505.
18. Lawn SD, Brooks SV, Kranzer K, Nicol MP, Whitelaw A, et al (2011) Screening for HIV-associated tuberculosis and rifampicin resistance before antiretroviral therapy using the Xpert MTB/RIF assay: a prospective study. PLoS Med 8: e1001067.
19. Scherer LC, Sperhacke RD, Jarczewski C, Cafrune PI, Michelon CT, et al (2011) Comparison of two laboratory-developed PCR methods for the diagnosis of pulmonary tuberculosis in Brazilian patients with and without HIV infection. BMC Pulm Med 11: 15.
20. Soini, H., and J. M. Musser. (2001). Molecular diagnosis of Mycobacteria. Clin. Chem. 47:809-814
21. Jain Amita, Tiwari Vandana RS. Guleria, Verma RK. (2002). Qualitative Evaluation of Mycobacterial DNA Extraction Protocols for Polymerase Chain Reaction. Molecular Biology Today 3: 43-50.
22. Singh JP, Verma R, Chaudhuri (2006) Random amplified polymorphic DNA (RAPD) analysis of Mycobacterium tuberculosis strains in India P - J. Vet. Sci. 7,181-187
23. Woods, G. L. (2001). Molecular techniques in mycobacterial detection. Pathol. Lab. Med. 125:122-126.
24. Querol JM, Farga MA, Granda D, Gimeno C, García-de-Lomas J et al., (1995). The utility of polymerase chain reaction (PCR) in the diagnosis of pulmonary tuberculosis. Chest; 107:1631-5.
25. Ulukanligil M, Aslan G, Tasci S. (2000). A comparative study on the different staining methods and number of specimens for the detection of acid fast bacilli. Mem Inst Oswaldo Cruz.; 95: 855-8.
26. Prasanthi K and Kumari AR.( 2005). Efficacy of fluorochrome stain in the diagnosis of pulmonary tuberculosis co-infected with HIV. Indian Journal of Medical Microbiology; 23(3):179-85.
27. Kulkarni Savita, Singh P., Memon Aafreen, Nataraj Gita, Kanade Swapna et al., (2012) An in-house multiplex PCR test for the detection of Mycobacterium tuberculosis, its validation & comparison with a single target TB-PCR kit. Indian J Med Res 135:788-794.
28. Tiwari Vandana, Jain Amita and Verma R. K. (2003) Application of enzyme amplified mycobacterial DNA detection in the diagnosis of pulmonary & extra-pulmonary tuberculosis. Indian J Med Res 118: 224-228.
29. Stella Sala Soares Lima, Wanessa Trindade Clemente, Moisés Palaci, Reinaldo Vieira Rosa et al. (2008) Conventional and molecular techniques in the diagnosis of pulmonary tuberculosis: a comparative study. J Bras Pneumol. 34(12):1056-1062.
DOI: http://dx.doi.org/10.18535/jmscr/v3i8.52
