Research Article

Role of Polymerase Chain Reaction in diagnosis of Tuberculosis as compared to routine tests

Dr Haris Memon, Dr Muhammad Haroon Mujtaba Memon, Dr Mahum Shahab, Dr Mahmood Iqbal, Dr Ghulam Murtaza Memon,
Article Date Published : 28 June 2019 | Page No.: 4490-4494 | Google Scholar


Download data is not yet available.


Introduction: There is a need for rapid and sensitive detection of Mycobacterium tuberculosis in clinical samples. A study was conducted in which the target for the amplification being a segment of IS6110 in the M. tuberculosis chromosome was evaluated using real time PCR and its results were compared with routine tests, using pulmonary and extra-pulmonary specimen.

Methods: In this descriptive cross-sectional retrospective study, specificity and sensitivity of PCR were analyzed. A total of 293 clinical samples were processed at a tertiary care hospital of Peshawar, during the time period of 2016-2018, from patients suspected of having pulmonary and extra-pulmonary tuberculosis and Follow up patients with DOTS treatment and MDR treatment that are referred by tertiary hospital were also included in this study after taking their informed consent. Patients not willing to participate in the study were excluded. For identification specimens were stained by Ziehl Neelsen staining (ZN), cultured on Lowenstein–Jensen (LJ) medium and then confirmed by PCR for the detection of Mycobacterium tuberculosis (MTB).

Results: Of the 293 samples, 165(56.3%) were from males and 128(43.7%) females. Mean age was 44 years (2-85 years). Specimen types included: CSF (30.4%), pleural fluid (4.1%), sputum (15%), urine (2.4%), synovial fluid (2.4%), other fluids (33.1%) and biopsies (12.6%). Only 3.1% of specimens were ZN-smear positive for (MTB). LJ culture identified 7.2% whereas PCR method detected (MTB) in 15% of the total specimens. Using PCR as gold standard, ZN microscopy correctly identified 20.5% of total (MTB) positive specimens and LJ culture detected 47.7%.Specimen types showed significant association with PCR test: 42.9% of synovial fluid samples and 41.7% of pleural fluid samples; 28.6% of urine samples were positive for (MTB) by PCR method. This indicates that PCR analysis of these specimens’ exhibit greater positivity rates for (MTB) as opposed to CSF and other fluids and biopsies

Conclusions: TB PCR is a rapid and reliable test in the diagnosis and management of tuberculosis.

Keywords: polymerase chain reaction, Mycobacterium tuberculosis, clinical evaluation


Tuberculosis is a global disease affecting one third of worldwide population. Every year 8 million new cases are diagnosed and of which 3 mill ion people dies .1 In America, from last two decade there is an increase in TB incidence number and it is increasing day by day 2 . P akistan is the 5th amongst 22 countries with highes t burden of TB 3 . In Pakistan, approximately 430,000 people of which 15,000 children get Tuberculosis (TB) and 70,000 deaths occurs every year due to the disease 3 . Without proper treatment, up to two thirds of people with TB will die 4 . Tuberculosis is a bacterial infection. Various species cause tuberculosis, among them mycobacteria is the most common specie 5 . Mycobacteria tuberculosis is an acid fast bacilli and is slow grow ing and facultative microbe 6 . Usually tuberculosis is diagnosed by routine tests like ZN smear chest X-rays and culture examination7 . These routine tests are not that effective due to low levels of mycobacterium and prolong time consuming procedures 8 . Of which, Z N smear is easy and simple test to perform; but it has low specification and sensitivity 9 . Mycobacteria Culture usually takes 4-6 week s to make diagnosis and it gives 20-30% of false negative results 10 . T his makes the diagnosis and treatment slow and compliance of TB patient difficulty which leads to emergence of MT drug resistance .11 There are various factors which increase TB incidence esp ecially when it co-exist with HIV as well as in low socioeconomic countries where it spreads easily and especially where facilities are insufficient 12 or in state of overcrowding and in hospital setting when diagnosis is either delayed or initiation of its treatment is delayed 13 . Even in developed countries where culture takes long, non-tuberculosis mycobacteria were detected. 14,15 For rapid and cost-effe ctive diagnosis of tuberculosis, new techniques are needed. 4 Polymerase chain reactions are the most suitable option for the diagnosis of MTB and PCR can even detect MTB in negative samples with high degree of sensitivity and specificity 16,17 , in both pulmonary and extra-pulmonary cases. PCR detects tuberculosis within 1-2 hrs with one sputum. Even in developing countries, PCR has a promising future in diagnosis of tuberculosis. 15 Previous studies showed that PCR sensitivity and specificity ranges from 77% to >95% of smear positive spemen 18,19 . Mycobacte ria DNA was prepared from microscopic slide 20 find out about the effectiveness and performance of PCR and its comparison with ZN microscopy and LJA culture was done in detecting (MTB).

materials and methods

This is descriptive cross-sectional retrospective study to compare sensitivity, specificity, positive predictive value (PPV) and neg ative predictive value (NPV) of Ziehl Neelsen (ZN) microscopy and Lowenstein Jensen (LJ) medium culture in detecting Mycobacterium tuberculosis ( (MTB)) and comparing with PCR assay as the gold standard.

A total of 293 samples of various specimen types were collected at main tertiary teaching hospital at Peshawar from patients suspected of having pulmonary and extra-pulmonary tuberculosis. Specimens underwent Ziehl Nee lsen staining, were cultured in LJ medium and processed by PCR for the detection of (MTB).

The data was analyzed using computer statistical package of social sciences (SPSS) version 22.0. Fisher exact test was used to determine statistical significance.


Of the 293 samples, 165(56.3%) were from males and 128(43.7%) were of females. Mean (± SD) age was 44 (±20) ranging from 2 years to 85 years. Specimen types included: CSF (n=89), pleural fluid (n=12), sputum (n=44), urine (n=7), synovial fluid (n=7), other flu ids (n=97) and biopsies (n=37).

Table 1
Gender Male 165(56.3%)
Female 128(43.7%)
ZN smear Positive 9(3.1%)
Negative 284(96.9)
LJ culture Positive 21(7.2%)
Negative 23(92.8%)
PCR Positive 44(15%)
Negative 249(85%)
Specimen types CSF 89(30.4%)
Pleural fluid 12(4.1%)
Sputum 44(15%)
Urine 7(2.4%)
Synovial fluid 7(2.4%)
Other fluids 97(33.1%)
Other biopsies 37(12.6%)

Only 3.1% (n=9) of specimens were ZN-smear positive for (MTB). LJ culture identified 7.2% (n=21) whereas PCR method detected (MTB) in 15% (n=44) of the total specimens. Using PCR as gold standard, ZN microscopy correctly identified 20.5% of total (MTB) positive specimens and LJ culture detected 47.7%.

Table 2
Sensitivity Specificity Positive predictive value Negative predictive value
PCR 100% 100% 100% 100%
ZN staining 20% 100% 100% 88%
LJ culture 47% 100% 100% 91%

Neither ZN microscopy nor LJ culture identified a truly negative patient as a positive patient therefore exhibit 100% specificity. Both tests did not give false positive results and thus exhibit 100% PPV. However ZN smearing failed to detect 34 true positive patients and LJ culture also failed to detect (MTB) in 23 specimens thus both have low sensitivity and low NPV as opposed to PCR method.

Fisher exact tests were performed to detect any association between gender, specimen type and laboratory test. There is no significant difference (P-value = 0.408) between males and females in the detection of (MTB) by PCR, LJ culture medium nor by ZN microscopy.

Specimen types showed significant association with PCR test: 42.9% of synovial fluid samples and 41.7% of pleural fluid samples; 28.6% of urine samples were positive for (MTB) by PCR method. This indicates PCR analysis of these specimens ’ exhibit greater positivity rates for (MTB) as opposed to CSF and other fluids and biopsies.

Table 3
PCR Result P value
Positive Negative
Specimen type CSF 12(13.5%) 77(86.5%) <0.005 (0.002)
Pleural fluid 5(41.7%) 7(58.3%)
Sputum 10(22.7%) 34(77.3%)
Urine 2(28.6%) 5(71.4%)
Synovial fluid 3(42.9%) 4(57.1%)
Other fluids 11(11.3%) 86(88.7%)
Other biopsies 1(2.7%) 36(97.3%)
Gender Male 26(15.8%) 139(84.2%) >0.05(0.408)
Female 18(14.1%) 110(85.9%)

Figure 1 :


Tuberculosis is an infectious disease caused b y Mycobacterium tuberculosis 21 .Tuberculosis is the second leading cause of death. Every year tuberculosis is effecting 10.4 million people around the world. In Unit ed States, during 1985-1992, an increase by 20% was reported in the incidence of tuberculosis to the centre for disease control cases. Since 1992 Tuberculosis, awareness became more and TB control became more manageable. 22,23,24,25,26,27

In Southeast, including Pakistan, Tuberculosis kills more people than any other infectious disease. Pakistan is 22nd among the countries according to world health Organization. Pakistan have high rate of MDR-TB and XDR-TB strains have been found in this Southeast Asia than any other infectious disease. According to World Hea lth Organization (WHO) reports, r apid TB control followed by adequate treatment are important in prevention of TB transmission and misdiagnosis28,29,30

In the local population where TB prevalence is high, our study has shown PCR performed better than the current routine diagnostic processes of ZN smear microscopy and LJ culture in detecting Mycobacteria tuberculosis in various specimen.

In this study, ZN showed least sensitivity (20%) of the 3 diagnostic methods concurrent with previous studies. From their research, Chakravorty et al found that the conventional smear method to have 3.9% sensitivity which was increased to 21.1% by universal sample processing technique 31 reported ZN smearing to have 50% sensitivity32,33 . This is due to ZN sensitivity being directly influenced by the HIV sta tus of the patient as reported by Lydia et al . 34 Moreover, the sensitivity of ZN is dependent on the type and quality of the specimen (34). T his is similar finding to our study as ZN mostly detected (MTB) in sputa as opposed to other specimens (p value <0.001). One of the reasons for low sensitivity is reported to be due to the fact that 104/ml is required for AFB to be seen using smear microscopy 35,36 .In this research, LJ culture method demonstrated sensitivity of 47%. Chakravrty et al reported that conventional culture detected zero cases of MTB but universal sample processing culture method demonstrated 7.9% sensitivity ( 31). I n the past (MTB) culture as a gold standard with estimated sensitivity and specificit y rates of 96% and 81%. However, a meta-analysis carried out in 2009, states (MTB) culture has limited value in clinical diagnosis as its sensitivity specificity rates have varied significantly from study to study. 37 A previous study in Pakistan reported a sensitivity rate of (MTB) culturing to be 15%-20% on over 50,000 specimens received from different geographical areas of the country. Our data revealed that PCR analysis showed 100% specificity and sensitivity38 . Bainomugisa et al showed PCR to have 100% sensitivity and 99% specificity 39 . A Study conducted in Lusaka, using low-cost in-house one –tube nested PCR, showed 55% of sensitivity 40,7,20 . Cheng et al reported TB PCR to have overall sensitivity of 78.3% and a specificity of 100 %41 .In our study PCR positivity rates were higher in specimens such as synovial fluid and pleural fluid as opposed to other specimens. This is a statistically significant with a p-value of < 0.005 (0.002). This may be because of larger volume of bodily fluid as opposed to that of sputa or other tissues specimens. This is similar finding to another study in Karachi, Pakistan where Amin et al. reports PCR assay to demonstrate positively rates of 70% in Bronchial Alveolar Lavage, Pleu ral fluid specimens 42 . This is concurrent with study by Chakravorty et al where PCR efficiencies were significantly high in samples of pleural fluid31 .

In Pakistan due to insufficient facilities it is difficult to control (MTB) transmission. However routine tests like ZN smear, culturing and PCR methods are used in diagnosis of TB. ZN staining is simple and fast test but has low sensitivity and specificity. Culturing tuberculosis has greater sensitivity but is time-consuming, it takes many weeks to give results. PCR facilitates prompt detection of infectious agent in various specimen types, thus is appropriate for both pulmonary and extra pulmonary tuberculosis


  1. World Health Organisation: WHO REPORT 2007. Global tuberculosis control. Surveillance, planning, financing .2007.
  2. Tuberculosis morbidity-United States Morb Mortal Wkly Rep.1994;44:395-395.
  3. Qasim (MTB) kills over 70,000 people every year in Pakistan. The News .2016;3.
  5. Advances in diagnosis of tuberculosis Butt T, Karamat K A, Ahmad R N, Mahmood A. Pak J Pathol.2001;12(1):1-3.
  6. Differential expression of 10 sigma factor genes in Mycobacterium tuberculosis Manganelli Riccardo, Dubnau Eugenie, Tyagi Sanjay, Kramer Fred Russell, Smith Issar. Molecular Microbiology.1999;31(2).
  7. Molecular techniques in mycobacterial detection Soini H, Musser J M. Pathol. Lab. Med.2001;47(7):122-126.
  8. Turnaround times for mycobacterial cultures Styrt B A, Shinnick T M, Ridderhof J C, Crawford J T, Tenover F C. J Clin Microbiol.1997;35:1041-1042.
  9. Tuberculosis transmission in the 1990 s Hamburg M A, Freiden T R. CrossRefPubMed Order article via Infotrieve Google Scholar.1994;332:1071-1076.
  10. New diagnostic tools for tuberculosis Perkins M D. Int. J. Tuberc. Lung Dis.2000;4:182-188.
  11. Increased tuberculosis burden due to demographic transition in Korea from Park Y K, Park Y S, Na K I, Cho E H, Shin S S, Kim H J. Tuberc Respir Dis.2001;74(11):104-110.
  12. Tuberculosis in Pakistan: Are We losing the battle Khan A. .2003;53.
  13. Global tuberculosis control .2002.
  14. Delayed diagnosis of pulmonary tuberculosis in city hospitals Mathur P, Sacks L, Auten G, Sall R, Levy C, Gordin F. Arch Intern Med.1994;154(14):306-310.
  15. Cost-effectiveness of the polymerase chain reaction versus smear-examination for the diagnosis of tuberculosis in Kenya: a theoretical model Roos B R, Van Cleeff M R A, Githui W A, Kivihya-Ndugga L, Kibuga D K, Klatser P R. Int. J. Tuberc. Lung Dis.1998;2(15):235-241.
  16. Molecular diagnosis of mycobacteria Soini H, Musser J M. Clin Chem.2001;47(16):809-823.
  17. Laboratory diagnosis of mycobacterial infections: new tools and lessons learned Hale Y M, Pfyffer G E, Salfinger M. Clin Infect Dis.2001;33(17):834-846.
  18. Assessment by meta-analysis of PCR for diagnosis of smear-negative pulmonary tuberculosis Sarmiento O L, Weigle K A, Weber Alexander J, Miller D J, W C. J Clin Microbiol.2003;41(18):3233-3240.
  19. Molecular detection of Mycobacterium tuberculosis: impact on patient care Kaul K L. Clin. Chem.2001;47:1553-1558.
  20. A comprehensive study on the efficiency of the routine tuberculosis diagnostic process in Nairobi Van Cleeff M R A, Kivihya-Ndugga L, Githui W A, Nganga L, Odhiambo J A, Klatser P R. Int. J. Tuberc. Lung Dis.2003;7:186-189.
  21. Effectiveness of qPCR for the Detection of TB .2017.
  22. Epidemiology of tuberculosis in the United States Cantwell M F, Snider D E, Cauthen G M, Onorato I M. JAMA.1985;272:535-539.
  23. Centers for Disease Control and Prevention. Nosocomial transmission of multidrug-resistant tuberculosis to health-care workers and HIV-infected patients in an urban hospital-Florida MMWR Morb Mortal Wkly Rep.1991;40(23):718-722.
  24. Centers for Disease Control and Prevention. Nosocomial transmission of multidrug-resistant tuberculosis among HIV-infected persons-Florida MMWR Morb Mortal Wkly Rep.1988;40(24):585-591.
  25. Centers for Disease Control and Prevention. Transmission of multidrug-resistant tuberculosis among immunocompromised persons in a correctional system MMWR Morb Mortal Wkly Rep.1992;41(25):507-509.
  26. Clinical presentation and outcome of patients with HIV infection and tuberculosis caused by multiple-drug-resistant bacilli Fischl M A, Daikos G L, Utamchandani R B. Ann Intern Med.1992;117(26):184-190.
  27. An outbreak of multidrug-resistant tuberculosis among hospitalized patients with the acquired immunodeficiency syndrome B R, Tokars J I, Grieco M H. N Engl J Med.1992;326(27):1414-1521.
  28. Global tuberculosis control. Surveillance, planning, financing WHO.2007;(28).
  29. Coninx R: Tuberculosis in complex emergencies Bull World Health Organ.2007;85(29).
  30. Trends in Mycobacterium tuberculosis resistance Hasan R, Jabeen K, Mehraj V, Zafar F, Malik F, Hassan Q. .1990;13:3-3.
  31. All Rights Reserved.Diagnosis of Extrapulmonary Tuberculosis by Smear, Culture, and PCR Using Universal Sample Processing Technology Lydia et al and Vignesh et al 31)Soumitesh Chakravorty, 1 Manas Kamal Sen, 2 and Jaya Sivaswami Tyagi1. Copyright 2005.2005;43:4357-4362.
  32. Comparison of PCR with the Routine Procedure for Diagnosis of Tuberculosis in a Population with High Prevalence of Tuberculosis and Human Immunodeficiency Virus.Lydia Kivihya-Ndugga Maarten Van Cleeff Ernest, Juma Joseph, Kimwomi Willie, Githui Linda, Oskam -. .2004;42:1012-1015.
  33. Value of single acid-fast bacilli sputum smears in the diagnosis of tuberculosis in HIV-positive subjects Ramachandran Vignesh, Pachamuthu Balakrishnan, Shankar Esaki Muthu Murugavel, Kailapuri Gangatharan Hanas, Settu Cecelia Anitha, Jebaraj Solomon, Suniti Kumarasamy, Nagalingeswaran -. J Med Microbiol.;(33):1709-1710.
  34. Comparison of PCR with the Routine Procedure for Diagnosis of Tuberculosis in a Population with High Prevalences of Tuberculosis and Human Immunodeficiency Virus 34) .Lydia Kivihya-Ndugga, 1 Maarten van Cleeff, 2 Ernest Juma, 1 Joseph Kimwomi, 1 Willie Githui, 1 Linda Oskam, 2 Anja Schuitema, 2 Dick van Soolingen, 3 Lucy Nganga, 1, 4 Daniel Kibuga, 5 Joseph Odhiambo, 1, 4 and Paul Klatser. J Clin Microbiol.2004;42(3):15004046-15004046.
  35. Impact of HIV infection in Zambia A cross sectional study Elliot A M, Luo N. BMJ.1990;301:412-415.
  36. Sensitivity of direct versus concentrated sputum smear microscopy in HIV-infected patients suspected of having pulmonary tuberculosis Adithya Cattamanchi, David Dowdy, Davis W, Lucian J, William Worodria, Samuel Yoo, Moses Joloba, John Matovu, Hopewell Philip, C, Laurence Huang. BMC Infectious Diseases.2009;9(36):53-53.
  37. Diagnostic Accuracy of In-House PCR for Pulmonary Tuberculosis in Smear-Positive Patients: Meta Analysis and Metaregression Greco S, Rulli M, Girardi E, Piersimoni C, Saltini C. J Clin Microbiol.2009;47:569-576.
  38. Fluoroquinolone-resistant Mycobacterium tuberculosis Jabeen K, Shakoor S, Chishti S, Ayaz A, Hasan R. .2005.
  39. Use of real time polymerase chain reaction for detection of M. tuberculosis, M. avium and M. kansasii from clinical specimens Bainomugisa A, Wampande E, Muchwa C. BMC Infect Dis.2015;15(39):181-181.
  40. Utility of nucleic acid amplification techniques for the diagnosis of pulmonary tuberculosis in sub-Saharan Africa Kambashi B, Mbulo G, Mcnerney R, Tembwe R, Kambashi A, Tihon V, Godfrey-Faussett P. Int. J. Tuberc. Lung Dis.2001;5:364-369.
  41. Clinical evaluation of the polymerase chain reaction for the rapid diagnosis of tuberculosis Cheng C C, Yam W C, Hung I F N, Woo P C Y, Lau S K P, Tang B S F, Yuen K Y. J Clin Pathol.2004;57(41):281-285.
  42. PCR could be a method of choice for identification of both pulmonary and extra-pulmonary tuberculosis Iram Amin, Muhammad Idrees. Zunaira Awan, Muhammad Shahid, Samia Afzal and Abrar Hussain.BMC Research Notes20114. BioMed Central Ltd.2010;(42).

Author's Affiliation

Copyrights & License

International Journal of Medical Science and Clinical invention, 2019.
Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

Article Details

Issue: Vol 6 No 06 (2019)
Page No.: 4490-4494
Section: Research Article

How to Cite

Memon, D. H., Haroon Mujtaba Memon, D. M., Shahab, D. M., Iqbal, D. M., & Murtaza Memon, D. G. (2019). Role of Polymerase Chain Reaction in diagnosis of Tuberculosis as compared to routine tests. International Journal of Medical Science and Clinical Invention, 6(06), 4490-4494.

Download Citation