IMPORTANCE OF POLYMERASE CHAIN REACTION IN DIAGNOSIS OF PULMONARY AND EXTRA-PULMONARY TUBERCULOSIS

Authors

  • Sajjad Iqbal
  • Rashid Ahmed
  • Saleem -uz-Zaman Adhami
  • Asim Mumtaz

Abstract

Background: 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.

References

World Health Organization. Global tuberculosis control:

epidemiology, strategy, financing. WHO report, 2009.

Available at: http://whqlibdoc.who.int/publications/2009/

_eng.pdf.

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

;16:569–601.

Chakravorty S, Kamal M, Tyagi JS. Diagnosis of Extrapulmonary tuberculosis by Smear, Culture and PCR, Using

Universal Sample Processing Technology. J Clin Microbiol

;43:4357–62.

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

;24:15–7.

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

Pathol 2000;53:355–61.

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

;75:960–3.

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

;124:81–8.

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

;31:2882–9.

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

;34:1209–15.

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

;34:834–41.

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

;18(2):25–8.

Maamari F. Case-finding tuberculosis patients: diagnostic and

treatment delays and their determinants. East Mediterr Health J

;14:531–45.

Published

2011-03-01

Most read articles by the same author(s)