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Revalidation of smear negative tubercular infection by conventional and by PCR applications

Kanchan Srivastava, Dinesh K. Tripathi, Kishore K. Srivastava, Surya Kant*

Abstract


IS6110 sequence based Polymerase Chain reaction (PCR) was compared with conventional bacteriological techniques in the laboratory diagnosis of Mycobacterium tuberculosis (MTB). A retrospective study involving one hundred and twenty six, non-repeated clinical isolates patients being investigated for tuberculosis. The samples were also processed for Ziehl-Neelsen (ZN) staining for acid fast bacilli (AFB) and culture for MTB. All the samples were processed for PCR amplification with primer targeting 123 bp fragments of insertion sequences IS6110 of M. tuberculosis complex (MTC) and the sensitivity of PCR was analyzed. Of the 126 patients, 100% and 97.3% were smears and culture positive for MTB respectively. Using culture as the gold standard, the overall sensitivity of PCR was 97.62%, and for either positive or either negative clinical isolates it was 97.06%% and 92.31%, respectively. The current study evaluated a PCR assay for the detection of MTC strains by targeting the IS6110 insertion element. This PCR has emerged as a rapid, reliable and a potent tool in establishing the diagnosis of tuberculosis with higher sensitivity than ZN microscopy and greater alacrity than culture.

Keywords


IS6110; M. tuberculosis; smear positive; culture positive; polymerase chain reaction

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References


WHO, Anti-tuberculosis drug resistance in the world. Report No. 2. Prevalence and trends. WHO/CDS/TB/2000.278, 2000

WHO Report, Global Tuberculosis Control Epidemiology, Strategy, Financing, 2009.

WHO, TB country Profile-Jordan. Source: www.who.int/tb/data, 2010.

Onyebujoh P, Rook GAW, Tuberculosis, Nat. Rev Microbiol, 2004, vol. 2, pp. 930–932.

Steinbrook R, Tuberculosis and HIV in India, N. Engl. J. Med, 2007, 356, 1198-1199.

Tostmann A, Kik SV, Kalisvaart NA, Tuberculosis transmission by patients with smear-negative pulmonary tuberculosis in a large cohort in the Netherlands, Clin Infect Dis, 2008, 47, 1135-1144.

Waard JH, Robledo J, Conventional Diagnostic Methods, Chapter 12. In: Tuberculosis, 1st Ed. Palomino JC, Leao SC, Ritacco V, Eds. 2007, pp. 401-24. Pulmonary tuberculosis, Indian J Med Microbiol, 2008, 26,352-355.

Pai MM, Steingart K, Ramsay A, New and improved tuberculosis diagnostics: evidence, policy, practice, and impact. Cur. Opinion in Pulmonary Medicine, 2010, 16, 1-14.

Amin I, Idrees M, Awan Z, Shahid M, Afzal S, Hussain A, PCR could be a method of choice for identification of both pulmonary and extra-pulmonary tuberculosis, BMC Research Notes, 2011, 4, 332.

Barani R, Sarangan G, Antony T, Periyasamy S, Kindo AJ, Srikanth P, Improved detection of Mycobacterium tuberculosis using two independent PCR targets in a tertiary care centre in south India, J Infect Dev Ctries, 2012, 6, 46-52.

Maurya AK, Kant S, Nag VL, Kushwaha RA, Kumar M, Dhole TN, Comparative evaluation of IS6110 PCR via conventional methods in rapid diagnosis of new and previously treated cases of extra pulmonary tuberculosis, Tuberc Toraks, 2011, 59, 213-220.

Sharma SK, Sethi S, Sharma M, Meharwal SK, Katoch VM, Jindal SK, Tewari R, Development and evaluation of a multiplex polymerase chain reaction for the detection of Mycobacterium tuberculosis from pulmonary specimens, Scand J Infect Dis.2012, 44, 739-744.

Venkataraman and Paramasivan CN, Bacteriological methods in laboratory diagnosis of Tuberculosis. Chetput, Chennai, India; Tuberculosis Research Center, 1999, ICMR.

Ganavalli S, Ajantha et al., PCR in EPTB www.jcdr.net J of Clinical and Diagnostic Research, 2013, 7, 1012-1015.

Foulds J, O'Brien R, New tools for the diagnosis of tuberculosis: the perspective of developing countries, Int J Tuberc Lung Dis, 1998, 2, 778–783.

Beige LJ, Schaberg T, Finckh U, Fischer M, Mauch H, Lode H, Köhler B and Rolfs A, Clinical evaluation of a Mycobacterium tuberculosis PCR assay, Clin Microbiol, 1995, 33, 90–95.

Brisson-Noel A, Aznar C, Chureau C, Nguyen S, Pierre C & other authors, Diagnosis of tuberculosis by DNA amplification in clinical practice evaluation, Lancet, 1999, 338, 364–366.

Gopinath K And Singh S, Multiplex PCR assay for simultaneous detection and differentiation of Mycobacterium tuberculosis, Mycobacterium avium complexes and other Mycobacterial species directly from clinical specimens, J Applied Microbiol, 2009, 107, 425-435.

Parandaman, Utility of PCR using 2 probes for rapid diagnosis of tubercular pleuritis in comparison to conventional methods, Ind.J. Med. Res, 2000, 112, 47-51.

Eisenach KD, Cave MD, Bates JH, Crawford JT, Polymerase chain reaction amplification of repetitive DNA sequence specific for Mycobacterium tuberculosis, J Infect Dis, 1990, 161, 977-981.

Chakravarty S, Kamal M, Tyagi JS, Diagnosis of Extra pulmonary Tuberculosis by smear, culture and PCR using universal samples processing technology, J Clin. Microbiol, 2005, 43, 4357-4362.

Negi SS, Khan SFB, Gupta S, Pasha ST, Khare S, Lal S, Diagnostic potential of IS6110, 38KDa, 65 Kda, and 85B sequence based polymerase chain reaction in the diagnosis of M.tuberculosis in clinical samples, Ind. J. Med. Microbial, 2007, 25, 43-49.

Sekar B, Selvaraj L, Alexis A, Ravi S, Arunagiri K, Rathinavel L, The Utility of IS6110 sequence based polymerase chain reaction in comparison to conventional methods in the diagnosis of Extra- pulmonary tuberculosis. Indian J Med Microbiol 2008, 26, 352-5.

Yam WC, Yuen KY, Seto WH, Direct detection of Mycobacterium tuberculosis in respiratory specimens using an automated DNA amplification assay and a single tube nested polymerase chain reaction (PCR), Clin Chem. Lab Med, 1998, 36, 597–9.

Brown TJ, Power EG, French GL, Evaluation of three commercial detection systems for Mycobacterium tuberculosis where clinical diagnosis is difficult, J Clin Pathol, 1999, 52, 193–197.

Almeda J, Garcia A, Gonzalez J. & other authors, Clinical evaluation of an in-house IS6110 polymerase chain reaction for diagnosis of tuberculosis, Eur J Clin Microbiol Infect Dis, 2000, 19, 859–867.

Bogard M, Vincelette J, Antinozzi R & other authors, Multicenter study of a commercial, automated polymerase chain reaction system for the rapid detection of Mtb in respiratory specimens in routine clinical practice, Eur J Clin Microbiol Infect Dis, 2001, 20, 724–731.

Rajalahti I, Vuorinen P, Liippo K & other authors, Evaluation of commercial DNA and rRNA amplification assays for assessment of treatment outcome in pulmonary tuberculosis patients, Eur J Clin Microbiol Infect Dis, 2001, 20, 746–750.

Takiff H, The molecular mechanisms of drug resistance in Mycobacterium tuberculosis. In: Bastian I, Portaels F, Eds. Multidrug-resistant tuberculosis. Dordrecht, the Netherlands, Kluwer Academic, 2000, 77–114.

Palomino JC, Nonconventional and new methods in the diagnosis of tuberculosis: feasibility and applicability in the field, ERJ, 2005, 26,339-350.

Kent PT, and Kubica GP, Public Health Mycobacteriology: A Guide for the level III laboratory. US Department of Health and Drug Resistance Pattern of Mtb Isolated from Patients Attending a Referral Hospital Wadud et al., Bangladesh, J Med Microbiol, 2000, 17, 1132-1140.

CDC. National plan for reliable tuberculosis laboratory services using a systems approach: recommendations from CDC and the Association of Public Health Laboratories Task Force on Tuberculosis Laboratory Services, MMWR, 2005, 54 (No. RR-6), 1-1.

Mazars E, Lesjean S, Banuls AL, Gilbert M, Vincent V, & other authors, High-resolution minisatellite-based typing as a portable approach to global analysis of Mycobacterium tuberculosis molecular epidemiology, PNAS, 2001, 98, 1901–1906.

Hosek P, Svastova M, Moravkova I, Pavlik MB, Methods of mycobacterial DNA isolation from different biological material, A review, Vet. Me, 2006, 51, 180-192.

Hill EB, Wayne LG, Gross M, Purification of mycobacterial deoxyribonucleic acid, J. bacterio, 1972, 112, 1033-1039.

Smittipat N and Palittapongarnpin P, Identification of possible loci of variable number of tandem repeats in Mycobacterium tuberculosis, Tuber. Lung. Dis, 2000, 80, 69-74.

Portillo-Gomez L, Morris SL, Panduro A, Rapid and efficient detection of extra-pulmonary Mycobacterium tuberculosis by PCR analysis, Int. J Tuberc Lung Dis, 2000, 4, 361–370.

Kearns AM, Freeman R, Steward M & other authors, A rapid polymerase chain reaction technique for detecting Mtb in a variety of clinical specimens, J Clin Pathol, 1998, 51, 922–924.

Agasino CB, Ponce de Leon A, Jasmer RM, Small PM, Epidemiology of Mtb. strains in San Francisco that does not contain IS6110, Int. J. Tuberc. Lung Dis, 1998, 29, 578-620.

Katoch VM, Newer diagnostic techniques for tuberculosis, Indian J. Med. Res, 2004, 120, 418–428.

Keservani RC, Pandey A, Misra A, Singh AK, Polymerase chain reaction (PCR): its comparison with conventional techniques for the diagnosis of extra pulmonary tubercular diseases, Indian J Surgery, 2004, 66, 84-88.

Negi SS Khan, SFB Gupta S Pasha, ST Khare, S Lal S. Protein antigen b (Pab) based PCR test in diagnosis of pulmonary& extra-pulmonary tuberculosis, Indian J Med Res, 2006, 124, 81–88.

Rathore M, Pai G, Jayalakshmi TK and Joshi DS, Rapid detection of multidrug resistant Mtb by RT-PCR based assay in Indian population. Recent Res. In Sci. and Tech, 2011, 3, 58-62.

Thierry D, Brisson-Noël A, Vincent-Lévy-Frébault V, Nguyen S, Guesdon JL, Gicquel B, Characterization of a Mycobacterium tuberculosis insertion sequence, IS6110, and its application in diagnosis, J Clin. Microbiol, 1990a, 28, 2668–2673.

Thierry D, Cave MD, Eisenach KD, Crawford JT, Bates JH and Gicquel B. IS6110, an IS-like element of Mycobacterium tuberculosis complex, Nucleic Acid Res, 1990b, 18, 188-192.

Metchock BG, Nolte FS, Wallace RJ Jr., In: Murray PR, Baron, EJ, Pfaller MA et al, 1999. Manual of clinical microbiology, 7th Ed. Washington DC: American Society for Microbiology 1999.




DOI: http://dx.doi.org/10.21746/ijbio.2015.02.004

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