Comparison of Polymerase Chain Reaction With Fluorescent Microscopy for Detecting Pulmonary Tuberculosis in Smear-Negative Sputum Samples in Quetta, Pakistan

Abstract

Background: One of the common clinical manifestations of extra-pulmonary tuberculosis is tuberculous meningitis (TBM). New methods, which are simple and effective, are necessary for early diagnosis of TBM. Objective: This study aimed to assess the use of fluorescence microscopy and polymerase chain reaction (PCR) techniques targeting pncA and IS6110. PCR is a fast and reliable technique for diagnosing tuberculosis (TB), particularly in paucibacillary specimens such as smear-negative sputum for diagnosing patients with Mycobacterium tuberculosis. Materials and Methods: The pncA and IS6110 multiplex-PCR methods were applied to analyze sputum using a specific pair of primers designed to amplify the insertion sequence, pncA, and IS6110 in the M. tuberculosis genome. A total of 200 sputum samples were collected from patients with TB. Out of 200 patients, 54.0% (n=108) and 46.0% (n=92) were males and females at Fatima Jinnah General and Chest Hospital, Quetta, respectively. Results: M. tuberculosis was detected as 15 (16.3%) and 18 (16.7%) for pncA and IS6110 using PCR and fluorescence microscopy, respectively. M. tuberculosis DNA detected by PCR and smear-negative fluorescence microscopy was 16.50% (n=33) and 14.70% (n=18), respectively. In other words, PCR assay detected the increased prevalence of M. tuberculosis. Risk factors revealed high exposure to receiving TB infection which was associated with urban areas (OR=1.07, 95% CI: 0.34–3.35), TB in family (OR=1.22, 95% CI: 0.38-3.88), over crowdedness (OR=2.93, 95% CI: 0.37-23.7), malnutrition (OR: 1.39; 95% CI: 0.52-3.74), and rural areas (OR: 1.54, 95% CI: 0.72-3.31). Conclusion: A wide range of molecular assays are now being developed and reviewed for detecting TB. We conclude that the use of pncA and IS6110 PCR assays is beneficial in the quick diagnosis of TB meningitis.