MOLECULAR DETECTION OF MYCOBACTERIUM TUBERCULOSIS IN SPUTUM SAMPLES USING REAL-TIME PCR AND MICROSCOPY

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a major global health concern, particularly in developing countries where diagnostic limitations delay treatment. Rapid and accurate detection is essential for early intervention and effective disease control. This study aimed to compare the diagnostic performance of Real-Time PCR (qPCR) with conventional Ziehl–Neelsen (ZN) microscopy for the detection of M. tuberculosis in sputum samples. A total of 100 sputum samples from clinically suspected pulmonary TB patients were analyzed. Each sample was examined microscopically for acid-fast bacilli (AFB) using the ZN staining method, followed by DNA extraction through the CTAB method and amplification of the IS6110 gene via qPCR. The diagnostic parameters—sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV)—were calculated, and agreement between both methods was assessed using Cohen’s kappa (κ) statistic. Microscopy detected 52 (52%) positive samples, while qPCR identified 68 (68%), including 16 additional cases that were missed by microscopy. Using qPCR as the reference standard, microscopy showed 76.4% sensitivity, 100% specificity, and an overall accuracy of 84%. The kappa value (κ = 0.72) indicated substantial agreement between the two methods (p < 0.001). The mean Ct value (27.8 ± 4.5) correlated inversely with bacterial load, confirming qPCR’s quantitative precision. Real-Time PCR proved to be a superior diagnostic tool, offering higher sensitivity, specificity, and rapid detection compared to conventional microscopy. Combining qPCR with microscopy can enhance diagnostic accuracy, enable early treatment initiation, and significantly strengthen tuberculosis control programs in line with the WHO End TB Strategy.