Lysis of tubercle bacilli in fresh and stored sputum specimens: implications for diagnosing tuberculosis in stored and paucibacillary specimens by PCR.
BACKGROUND
Nucleic 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.


RESULTS
Eighty-seven smear positive sputa from tuberculosis patients were analysed immediately and after storage at -20 degrees 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.


CONCLUSIONS
We 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.
Pathak
Divya
D
Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India. divya7pathak@rediffmail.com
Chakravorty
Soumitesh
S
Hanif
Mahmud
M
Tyagi
Jaya Sivaswami
JS
D001433:Bacteriolysis; D004269:DNA, Bacterial / Q000235:genetics*; D005615:Freezing; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods; D013048:Specimen Handling; D013183:Sputum / Q000382:microbiology*; D013997:Time Factors; D014376:Tuberculosis / Q000175:diagnosis*