Abstract
BackgroundNucleic acid amplification techniques are being used increasingly in diagnosing tuberculosis. In developing countries clinical samples are often stored for subsequent analysis since molecular tests are conducted at only a limited number of laboratories. This study was conducted to assess the speed at which mycobacteria undergo autolysis and free DNA is detected in the supernatant during low-temperature storage.ResultsEighty-seven smear positive sputa from tuberculosis patients were analysed immediately and after storage at -20°C. Timelines of 1 and 2 months were selected to assess the maximum extent of DNA loss that occurred during storage. All samples remained PCR- and smear-positive at 1 month and only 1 sample turned negative after 2 months. Bacterial lysis in the specimens was demonstrated by PCR analysis of supernatant fractions; 53% of the freshly analysed samples contained mycobacterial DNA in supernatants. PCR positivity increased significantly during storage (to 69% and 77% after 1 and 2 months of storage, respectively, P < 0.0001). Storage-associated bacterial lysis was accompanied by a decrease in smear grade status in 28 of 87 samples (P < 0.0001 after 2 months of storage) and a significant storage-associated reduction in bacterial numbers in the remaining samples.ConclusionWe conclude that (i) freshly isolated sputum contains both intact and lysed mycobacteria, (ii) lysis increased during storage and (iii) supernatant fractions routinely discarded during sample processing contain mycobacterial DNA. We propose that supernatant is a valuable sample for PCR for both fresh and stored specimens, particularly those with a low bacterial load in addition to conventional sediment.
Highlights
Nucleic acid amplification techniques are being used increasingly in diagnosing tuberculosis
In a preliminary study conducted to assess the impact of sputum storage on PCR, maximum bacterial autolysis in untreated samples was noted during room temperature vs. 4°C or -20°C storage (Dudeja M, unpublished observations)
The present study was designed to assess the effect of storage at -20°C of N-acetyl Lcysteine (NALC)-treated sputum on bacterial lysis
Summary
Nucleic acid amplification techniques are being used increasingly in diagnosing tuberculosis. Nucleic acid amplification technologies are being intensively assessed in diagnosing extrapulmonary TB owing to their speed, specificity and sensitivity [4] Their application is limited to sophisticated laboratories in developing countries including India and samples are often stored prior to analysis. The inherent delay in obtaining culture results is a serious limitation of the gold standard; it was proposed that when smear microscopy and DNA amplification were both positive, the diagnosis of active TB could be considered established [7]. For these various reasons PCR is being increasingly considered as an adjunct test for diagnosing TB in resource-poor settings
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