IMPORTANCE OF POLYMERASE CHAIN REACTION IN DIAGNOSIS OF PULMONARY AND EXTRA-PULMONARY TUBERCULOSIS
AbstractBackground: Pakistan ranks 8th on the list of 22 high-burden tuberculosis (TB) countries in the worldaccording to the World Health Organization’s (WHO) Global Tuberculosis Control 2009. Includingother reasons the main cause is improper and late diagnosis of the disease. PCR may play an importantrole to control the disease with its rapid, sensitive and specific diagnosis. But in Pakistan due to lake ofknowledge about this latest technique we are not using this technique appropriately. Clinicians still truston conventional methods of TB diagnosis, which are time consuming or insensitive. The present studywas arranged to highlight the importance of PCR in TB diagnosis in pulmonary and extra-pulmonarycases and its comparison with conventional methods. Methods: Samples obtained from 290 patients ofsuspected TB (pulmonary or extra-pulmonary) were subjected to ZN smear examination, LJ mediumculture and PCR test by amplifying 541bp fragment of Mycobacterium tuberculosis complex genome.The present prospective study is performed at Shalamar Hospital Lahore from November 2008 toNovember 2010. Results: A distinctly difference was observed in the test results done by PCR andother conventional techniques in pulmonary or extra-pulmonary tuberculosis samples (p<0.001). Thesensitivity of different tests was 68.62% for PCR, 26.90% for LJ medium culture, and 14.14% for ZNsmear examination (p<0.05). However, there was no significant difference between different tests as foras specificity was concerned. PCR test sensitivity in pulmonary and extra-pulmonary clinical sampleswas 78.34 and 61.76% respectively, being significantly higher (p<0.05) when compared withsensitivity of other tests. The mean detection time for M. tuberculosis was 25 days by LJ mediumculture and less than 1 day by smear examination and PCR test. Conclusion: PCR test is more sensitivethan ZN smear examination and LJ medium culture for the diagnosis of TB in pulmonary and extrapulmonary clinical samples.Keywords: Mycobacterium tuberculosis complex, Polymerase chain reaction, LJ medium culture, ZNstaining.
World Health Organization. Global tuberculosis control:
epidemiology, strategy, financing. WHO report, 2009.
Available at: http://whqlibdoc.who.int/publications/2009/
Dye C, Scheele S, Dolin P, Pathania V, Raviglione M. Global
burden of tuberculosis: Estimated incidence, prevalence, and
mortality by country. JAMA 1999;282:677–86.
Iqbal S, Iqbal R, Khan M, Akhter A, Shabbir I. Comparison of
two conventional techniques used for the Diagnosis of
Tuberculosis Cases. Int J Agri Biol 2003;5:545–7.
Montenegro SH, Gilman RH, Sheen P, Cama R, Cavides L,
Oberhelman RA. Improved detection of M. tuberculosis in
Peruvian children by use of heminested IS6110 PCR assay. Clin
Infec Dis 2003;36:6–23.
Witebsky FG, Kruczak-Filipov P. Identification of
Mycobacteria by conventional methods. Clin Lab Med
Chakravorty S, Kamal M, Tyagi JS. Diagnosis of Extrapulmonary tuberculosis by Smear, Culture and PCR, Using
Universal Sample Processing Technology. J Clin Microbiol
Kolk AH, Kox LF, van Leeuwen J, Kuijper S, Jansen HM.
Clinical utility of the polymerase chain reaction in the diagnosis
of extrapulmonary tuberculosis. Eur Respir J 1998;11:1222–6.
Dar L, Sharma SK, Bhanu NV, Broor S, Chakraborty M, Pande
JN, et al. Diagnosis of pulmonary tuberculosis by polymerase
chain reaction for MPT64 gene: an evaluation in a blind study.
Indian J Chest Dis Allied Sci 1998;40:5–16.
Rau N, Libman M. Laboratory implementation of the
polymerase chain reaction for the confirmation of pulmonary
tuberculosis. Eur J Clin Microbiol Infect Dis 1999;18:35–41.
Baron JE, Peterson LR, Finegold SM. Mycobacteria. In: Bailey
and Scott’s diagnostic microbiology. St. Louis, Missouri:
Mosby; 1994. p. 590–633.
Siddiqi SH. BACTEC 460 TB system. Product and procedure
manual. Maryland, USA: 1996 p. III 1-III 9.
Afghani B, Stutman HR. Diagnosis of Tuberculosis: can
polymerase chain reaction replace AFB smear and culture. J
Infect Dis 1995;172:903–4.
Amato RFD, Hochstein LH, Colaninno PM, Scardamaglia M,
Kim K, Mastellone AJ, et al.. Application of the Roche
Amplicor Mycobacterium tuberculosis (PCR) test to specimens
other than respiratory secretion. Dia Microbiol Infect Dis
Dagmar R, Tyagi S, Kappe R. Performance of a IS6110 based
PCR assay and the COBAS amplicor MTB PCR system for
detection of M. tuberculosis complex DNA in human lymph
node samples. J Clin Microbiol 2002;40:3089–92.
Singh KK, Muralidhar M, Kumar A, Chattopadhyaya TK,
Kapila K, Singh MK,, et al.. Comparison of inhouse polymerase
chain reaction with conventional techniques for the detection of
Mtuberculosis DNA in granulomatous lymphadenopathy. J Clin
Singh UB, Bhanu NV, Suresh VN, Arora J, Rana T, Seth P.
Utility of polymerase chain reaction in diagnosis of tuberculosis
from samples of bone marrow aspirate. Am J Trop Med Hyg
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
Clarridge JE, Shawar RM, Shinnick TM, Plikaytis BB. Largescale use of polymerase chain reaction for detection of
Mycobacterium tuberculosis in a routine mycobacteriology
laboratory. J Clin. Microbiol 1993;31:2049–56.
Honore-Bouakline S, Vincensini JP, Giacuzzo V, Lagrange PH,
Herrmann JL. Rapid diagnosis of extrapulmonary tuberculosis
by PCR: impact of sample preparation and DNA extraction. J
Clin. Microbiol 2003; 41:2323–9.
Kirschner P, Springer B, Vogel U, Meier A, Wrede A,
Kiekenbeck M, et al. Genotypic identification of mycobacteria
by nucleic acid sequence determination: report of a 2-year
experience in a clinical laboratory. J Clin Microbiol
Mangiapan G, Vokurka M, Schouls L, Cadranel J, Lecossier D,
Embden JV, et al. Sequence capture-PCR improves detection of
mycobacterial DNA in clinical specimens. J Clin Microbiol
Pfyffer GE, Kissling P, Jahn EM, Welscher HM, Salfinger M,
Weber R. Diagnostic performance of amplified Mycobacterium
tuberculosis direct test with cerebrospinal fluid, other
nonrespiratory, and respiratory specimens. J Clin Microbiol
Khan MA, Mirza SH, Abbasi SH, Butt T, Anwar M. Peripheral
blood-based polymerase chain reaction in diagnosis of
pulmonary tuberculosis. J Ayub Med Coll Abbottabad
Maamari F. Case-finding tuberculosis patients: diagnostic and
treatment delays and their determinants. East Mediterr Health J