Utility of Polymerase Chain Reaction in Diagnosis of Tuberculosis in Our Setup: A Ten Years Experience

Objective: To evaluate the yield of polymerase chain reaction (PCR) for detection of Mycobacterium tuberculosis from different clinical specimens. Study Design: Observational study. Place and Duration of Study: Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi, from January 2001 to December 2010. Methodology: Different clinical specimens received for Mycobacterium tuberculosis PCR were dealt during the study period. Contaminated samples like sputum were processed by the standard N-acetyl L-cysteine (NALC)-NaOH method. PCR protocols were followed as per manufacturer's manual. PCR was performed using a Thermal Cycler (Master Cycler, Eppendorf, Germany): an initial denaturation step at 94 degrees C for 3 minutes was followed by 40 cycles of denaturation at 94 degrees C for 30 seconds, annealing at 60 degrees C for 30 seconds and extension at 72 degrees C for 30 seconds and a final extension at 72 degrees C for 7 minutes. The products were held at 4 degrees C and later run on 1% agarose gel, stained with Ethidium bromide and visualized in ultraviolet (UV) transilluminator. Results: Out of a total 4620 samples for PCR, 299 were positive for Mycobacterium tuberculosis (6.5%). The percentage of samples from male patients were 63.2%. The mean age of patients was 38+/-11.5 years. Blood was the most frequent specimen received for PCR (46.66%), followed by body fluids (18.41%) and CSF (10.64%). Yield for different clinical samples was 63/471 for sputum (13.4%), 3/29 for endobronchial washings (10.3%), 59/851 for body fluids (6.9%) and 24/400 for urine (6%). Positive yield from blood was the lowest (101/2156, 4.7%). Conclusion: PCR for Mycobacterium tuberculosis is a rapid and reliable method for the diagnosis of both pulmonary and extrapulmonary tuberculosis. The highest positive yield was obtained from sputum and lowest from blood specimens.