Biosafety evaluation of the DNA extraction protocol for Mycobacterium tuberculosis complex species, as implemented at the Instituto Nacional de Salud, Colombia
Keywords:
Mycobacterium tuberculosis, exposure to biological agents, DNA, laboratory techniques, DNA procedures.
Abstract
Introduction. Manipulating Mycobacterium tuberculosis clinical specimens and cultures represents a risk factor for laboratory personnel. One of the processes that requires high concentrations of microorganisms is DNA extraction for molecular procedures. Pulmonary tuberculosis cases have occurred among professionals in charge of molecular procedures that require manipulation of massive quantities of microorganisms. This has prompted research studies on biosafety aspects of extraction protocols; however, as yet, no consensus has been reached regarding risks associated with the process.Objective. The biosafety was evaluated for the DNA extraction protocol of van Soolingen, et al. 2002 by determining M. tuberculosis viability at each process stage.
Materials and methods. Eight hundred eighty cultures were grown from 220 M. tuberculosis clinical isolates that had been processed through the first three DNA extraction stages. Molecular identifications of positive cultures used a PCR isolation of a fragment of the heat shock protein PRA-hsp65 and examination of its restriction enzyme profile (spoligotyping).
Results. Growth was seen in one culture with one of the procedures used. The molecular characterization did not correspond to the initially analyzed isolate, and therefore was deduced to be the product of a cross-contamination.
Conclusion. The DNA extraction protocol, as described by van Soolingen, et al. 2002 and as implemented at the Instituto Nacional de Salud, was established to be safe for laboratory personnel as well as for the environment.
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References
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13. Van Soolingen D, de Hass P, Kremer K. Restriction fragment length polymorphism (RFLP) typing of Mycobacteria. Bilthoven: National Institute of Public Health and the Environment; 2002.
14. Zwadyk P Jr, Down J, Myers N, Dey M. Rendering of mycobacteria safe for molecular diagnostic studies and development of a lysis method for strand displacement amplification and PCR. J Clin Microbiol. 1994;32:2140-6.
15. Collins C, Kennedy D. Laboratory-acquired infections: History, incidence, causes and prevention. 4th edition. Oxford: Butterworth-Heinemann; 1999.
16. Blackwood K, Burdz T, Turenne C, Sharma M, Kabani A, Wolfe J. Viability testing of material derived from Mycobacterium tuberculosis prior to removal from a containment level-III laboratory as a part of a laboratory risk assessment program. BMC Infect Dis. 2005;5:1-7.
17. Doig C, Seagar A, Watt B, Forbes K. The efficacy of the heat killing of Mycobacterium tuberculosis. J Clin Pathol. 2002;55:778-9.
18. Warren R, Kock M, Engelke E, Myburgh R, Van Pittius N, Victor T, et al. Safe Mycobacterium tuberculosis DNA extraction method that does not compromise integrity. J Clin Microbiol. 2006;44:254-6.
19. Djelouagji Z, Drancourt M. Inactivation of cultured Mycobacterium tuberculosis organisms prior to DNA extraction. J Clin Microbiol. 2006;44:1594-5.
20. Glynn J, Yates M, Crampin A. DNA fingerprint changes in tuberculosis: reinfection, evolution, or laboratory error? J Infect Dis. 2004;190:1158-66.
21. Small P, McClenny N, Singh S, Schoolnik G, Tompkins L, Mickelsen P. Molecular strain typing of Mycobacterium tuberculosis to confirm cross-contamination in the mycobacteriology laboratory and modification of procedures to minimize occurrence of false-positive cultures. J Clin Microbiol. 1993;31:1677-82.
22. Wurtz R, Demarais P, Trainor W. Specimen contamination in mycobacteriology laboratory detected by pseudo-outbreak of multidrug-resistant tuberculosis: analysis by routine epidemiology and confirmation by molecular technique. J Clin Microbiol. 1996;34:1017-9.
23. Burman W, Stone B, Reves R. The incidence of false-positive cultures for Mycobacterium tuberculosis. Am J Respir Crit Care Med. 1997;155:321-6.
24. Bhattacharya M, Dietrich S, Mosher L. Cross contamination of specimens with Mycobacterium tuberculosis: clinical significance, causes, and prevention. Am J Clin Pathol. 1998;109:324-30.
2. Bentwich Z, Maartens G, Torten D, Lal A, Lal R. Concurrent infections and HIV pathogenesis. AIDS. 2000;14:2071-81.
3. World Health Organization. Guidelines for the program-matic management of drug-resistant tuberculosis. WHO/HTM/TB/2006.361. Geneva: World Health Organization; 2006.
4. Broekmans J. Control strategies and programme management. In: Porter JD, McAdam PW, editors. Tuberculosis, back to the future. New York, N.Y: John Wiley & Sons Inc.; 1994. p. 171-92.
5. Supply P, Mazars E, Lesjean S, Vincent V, Gicquel B, Locht C. Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome. Mol Microbiol. 2000;36:762-71.
6. Devallois A, Goh K, Rastogi N. Rapid identification of mycobacteria to species level by PCR-restriction fragment length polymorphism analysis of the hsp65 gene and proposition of an algorithm to differentiate 34 myco-bacterial species. J Clin Microbiol. 1997;35:2969-73.
7. Van Embden J, Cave M, Crawford J, Dale J, Eisenach K, Gicquel B. Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: Recommendations for a standardized methodology. J Clin Microbiol. 1993;31:406-9.
8. Kamerbeek J, Schouls L, Kolk A, van Agterveld M, van Soolingen D, Kuijper S, et al. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol. 1997;35:907-14.
9. Menzies D, Fanning A, Yuan L, Fitzgerald M. Tuberculosis among health care workers. N Engl J Med. 1995;332:92-8.
10. Bemer-Melchior P, Drugeon H. Inactivation of Mycobacterium tuberculosis for DNA typing analysis. J Clin Microbiol. 1999;37:2350-1.
11. Somerville W, Thibert L, Schwartzman K, Behr M. Extraction of Mycobacterium tuberculosis DNA: a question of containment. J Clin Microbiol. 2005;43:2996-7.
12. Van Soolingen D, Hermans P, de Haas P, Soll D, van Embden J. The occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J Clin Microbiol. 1991;29:2578-86.
13. Van Soolingen D, de Hass P, Kremer K. Restriction fragment length polymorphism (RFLP) typing of Mycobacteria. Bilthoven: National Institute of Public Health and the Environment; 2002.
14. Zwadyk P Jr, Down J, Myers N, Dey M. Rendering of mycobacteria safe for molecular diagnostic studies and development of a lysis method for strand displacement amplification and PCR. J Clin Microbiol. 1994;32:2140-6.
15. Collins C, Kennedy D. Laboratory-acquired infections: History, incidence, causes and prevention. 4th edition. Oxford: Butterworth-Heinemann; 1999.
16. Blackwood K, Burdz T, Turenne C, Sharma M, Kabani A, Wolfe J. Viability testing of material derived from Mycobacterium tuberculosis prior to removal from a containment level-III laboratory as a part of a laboratory risk assessment program. BMC Infect Dis. 2005;5:1-7.
17. Doig C, Seagar A, Watt B, Forbes K. The efficacy of the heat killing of Mycobacterium tuberculosis. J Clin Pathol. 2002;55:778-9.
18. Warren R, Kock M, Engelke E, Myburgh R, Van Pittius N, Victor T, et al. Safe Mycobacterium tuberculosis DNA extraction method that does not compromise integrity. J Clin Microbiol. 2006;44:254-6.
19. Djelouagji Z, Drancourt M. Inactivation of cultured Mycobacterium tuberculosis organisms prior to DNA extraction. J Clin Microbiol. 2006;44:1594-5.
20. Glynn J, Yates M, Crampin A. DNA fingerprint changes in tuberculosis: reinfection, evolution, or laboratory error? J Infect Dis. 2004;190:1158-66.
21. Small P, McClenny N, Singh S, Schoolnik G, Tompkins L, Mickelsen P. Molecular strain typing of Mycobacterium tuberculosis to confirm cross-contamination in the mycobacteriology laboratory and modification of procedures to minimize occurrence of false-positive cultures. J Clin Microbiol. 1993;31:1677-82.
22. Wurtz R, Demarais P, Trainor W. Specimen contamination in mycobacteriology laboratory detected by pseudo-outbreak of multidrug-resistant tuberculosis: analysis by routine epidemiology and confirmation by molecular technique. J Clin Microbiol. 1996;34:1017-9.
23. Burman W, Stone B, Reves R. The incidence of false-positive cultures for Mycobacterium tuberculosis. Am J Respir Crit Care Med. 1997;155:321-6.
24. Bhattacharya M, Dietrich S, Mosher L. Cross contamination of specimens with Mycobacterium tuberculosis: clinical significance, causes, and prevention. Am J Clin Pathol. 1998;109:324-30.
How to Cite
1.
Ribón W, Castro C, González L, Rozo JC, Puerto G. Biosafety evaluation of the DNA extraction protocol for Mycobacterium tuberculosis complex species, as implemented at the Instituto Nacional de Salud, Colombia. Biomed. [Internet]. 2009 Dec. 1 [cited 2025 Apr. 4];29(4):561-6. Available from: https://revistabiomedicaorg.biteca.online/index.php/biomedica/article/view/133
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Published
2009-12-01
Issue
Section
Original articles
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