Rapid and specific detection of Mycobacterium tuberculosis directly from sputum specimens using IS6110 and pncA through multiplex-PCR

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Muhammad Rizwan Muhammad Arif Awan Muhammad Naeem Hamida Haider Abdul Samad, Muhammad Shafee, Safiullah Khan Achakzai Farah Sabeen Bugti1, Zafar Ahamad Sania Ashraf


Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis complex, is the second leading cause of death. One third of the world’s population is infected with TB. Every day more than 23000 people develop active TB and nearly 5000 die. Approximately 20-50% of patients with TB are smear negative, accounting for as much as 17% of TB transmission, posing an important public health hazard. In the objective of improved TB diagnosis and rapid detection of M. tuberculosis complex species particularly from paucibacillary sputum specimens, the sensitivity of Polymerase Chain Reaction (PCR) using IS6110 and pncA as genetic targets was compared with the conventional fluorescent microscopy. The study also aimed to check the sensitivity of two different genetic markers IS6110 and pncA for the detection of M. tuberculosis. Sputum specimens were collected from TB suspected subjects (N= 200; males=108; females=92; mean age=47±18.4) attending outpatient department at Fatima Jinnah General and Chest Hospital, Quetta. All the samples were decontaminated using N-acetyl-L-cysteine (NALC)-NaOH method and were subjected to auramine-O fluorescent microscopy and multiplex PCR using TB1 and TB2 primers specific for M. tuberculosis complex and pncATB-1.2 and pncAMT-2 primers specific to M. tuberculosis. Out of 200 specimens, M. tuberculosis around 15 (7.5%) was detected by fluorescent microscopy and 33 (16.5%) were detected through PCR, respectively. Whereas 18 smear negative specimens were found to be positive by PCR. Statistically significant difference between fluorescent microscopy and PCR was observed (p<0.001). Among these 33 positive cases, 18 (16.6%) were males and 15 (16.30%) were females with statistically no significant difference (OR: 1.03, 95% CI: 0.48-2.17; p>0.05). Similarly, age was not found to be associated (p>0.05) with the increased risk of TB, however, the disease incidence was more prevalent (20.89%) in patients aged 41–60 years. Multiplex PCR showed increased sensitivity as compared to fluorescent microscopy for TB diagnosis and will bea useful tool in detecting smears negative TB. Further, it can also be concluded that both genetic markers IS6110 and pncA showed similar sensitivity in the detection of M. tuberculosis.

Keywords: Quetta; Tuberculosis; Paucibacillary; Diagnosis; Genetic markers


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