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

Muhammad Rizwan

doi:10.19045/bspab.2017.60052

Abstract

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 0.05). 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 http://dx.doi.org/10.19045/bspab.2017.60052

Highlights

Introduction die of the diseaseOf these (420,000), Tuberculosis is an ancient, extensively around 175,000 cases are sputum smear prevalent, and more often deadly, positive and 9000 are the cases of MDR TB contagious disease caused by M.while remaining of the cases are “sputum tuberculosis complex species, usually M.smear negative TB” [5].tuberculosis [1]
The sputum specimens were analyzed for the detection of M. tuberculosis through the conventional fluorescent microscopy and the Polymerase Chain Reaction (PCR) targeting IS6110 and pncA
All 15 smear-positive sputum specimens were positive by PCR for M. tuberculosis

Summary

Introduction

Introduction die of the diseaseOf these (420,000), Tuberculosis is an ancient, extensively around 175,000 cases are sputum smear prevalent, and more often deadly, positive and 9000 are the cases of MDR TB contagious disease caused by M.while remaining of the cases are “sputum tuberculosis complex species, usually M.smear negative TB” [5].tuberculosis [1]. The throughout centuries and was titled smear microscopy is rapid and an “consumption” and “white plague” in the inexpensive method. It was world’s major cause of sensitivity and specificity with death from all causes, responsible for one smear negative cases [6]. TB take six to eight weeks due to the slow ranks 2nd major cause of death after HIV by growth of M. tuberculosis on solid media, an infectious disease and WHO estimates delaying time to results, followed by that some 2 billion individuals, 1/3rd identification with use of biochemical or population of the world, are infected with M

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Conclusion