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°C for 3 minutes was followed by 40 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds and extension at 72°C for 30 seconds and a final extension at 72°C for 7 minutes. The products were held at 4°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.