Molecular Diagnosis & Therapy

, Volume 14, Issue 4, pp 223–227 | Cite as

Comparative Evaluation of Nested PCR and Conventional Smear Methods for the Detection of Mycobacterium tuberculosis in Sputum Samples

  • Sathish Sankar
  • Babu Balakrishnan
  • Balaji Nandagopal
  • Kannaki Thangaraju
  • Subapriya Natarajan
Original Research Article


Background: With 1.8 million new cases each year, India carries 20% of the global burden of tuberculosis, a situation that is now further exacerbated with the emergence of drug resistance. The current diagnostic technique suggested by the Government of India’s Revised National Tuberculosis Control Programme is Ziehl-Neelsen staining of a sputum smear. This technique is known to be inadequate.

Objective: The aim of this study was to evaluate nested PCR (nPCR) in the detection of pulmonary tuberculosis in sputum samples in comparison with conventional smear findings, in an effort to improve detection rates from those obtained by the smear-alone approach.

Study Design: Patients attending a tertiary-care hospital (situated in a rural area of Vellore district) with clinical suspicion of pulmonary tuberculosis were prospectively recruited from mid-April 2009 to mid-December 2009 and investigated. The sputum samples were stained by Ziehl-Neelsen staining for smear examination. DNA extracted from concentrated sputum was tested by nPCR, targeting the IS6110 sequence in the Mycobacterium tuberculosis genome.

Results: Among 84 patients tested (median age 45.5 years), 80.95% were from the rural community and 19.05% were from the peri-urban community. Seventeen patients (20.24%; mid-p 95% CI 31.5, 52.4) tested positive by the smear examination and 35 (41.67%; mid-p 95% CI 12.7, 29.8) tested positive by nPCR. The difference in detection rates was statistically significant (χ2 = 9.02; p = 0.002). The κ coefficient between smear findings and nPCR findings was 0.47, which was a statistically significant agreement (Z = 4.91; p<0.0001).

Conclusion: This report describes the molecular detection of M. tuberculosis in patients’ sputum samples tested by the nPCR format, using IS6110 as a target sequence. A high prevalence of pulmonary tuberculosis was identified by the nPCR assay, which was shown to have a significantly higher detection rate than conventional smear staining.


Tuberculosis Pulmonary Tuberculosis Nest Polymerase Chain Reaction Sputum Sample Sputum Smear 
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The research work was funded by an intramural research support fund, which is gratefully acknowledged. The authors have no conflicts of interest that are directly relevant to the content of this study.


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© Adis Data Information BV 2010

Authors and Affiliations

  • Sathish Sankar
    • 1
  • Babu Balakrishnan
    • 1
  • Balaji Nandagopal
    • 1
  • Kannaki Thangaraju
    • 2
  • Subapriya Natarajan
    • 3
  1. 1.Division of Biomedical ResearchSri Narayani Hospital and Research CentreThirumalaikodi, Sripuram, VelloreIndia
  2. 2.Department of Community HealthSri Narayani Hospital and Research CentreThirumalaikodi, Sripuram, VelloreIndia
  3. 3.Department of General MedicineSri Narayani Hospital and Research CentreThirumalaikodi, Sripuram, VelloreIndia

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