title: Viability testing of material derived from Mycobacterium tuberculosis prior to removal from a containment level-III laboratory as part of a Laboratory Risk Assessment Program.
abstract: BACKGROUND
  In the field of clinical mycobacteriology, Mycobacterium tuberculosis (MTB) can be a difficult organism to manipulate due to the restrictive environment of a containment level 3 (CL3) laboratory. Tests for rapid diagnostic work involving smears and molecular methods do not require CL3 practices after the organism has been rendered non-viable. While it has been assumed that after organism deactivation these techniques can be performed outside of a CL3, no conclusive study has consistently confirmed that the organisms are noninfectious after the theoretical 'deactivation' steps. Previous studies have shown that initial steps (such as heating/chemical fixation) may not consistently kill MTB organisms.
  
  
  METHODS
  An inclusive viability study (n = 226) was undertaken to determine at which point handling of culture extraction materials does not necessitate a CL3 environment. Four different laboratory protocols tested for viability included: standard DNA extractions for IS6110 fingerprinting, crude DNA preparations for PCR by boiling and mechanical lysis, protein extractions, and smear preparations. For each protocol, laboratory staff planted a proportion of the resulting material to Bactec 12B medium that was observed for growth for 8 weeks.
  
  
  RESULTS
  Of the 208 isolates initially tested, 21 samples grew within the 8-week period. Sixteen (7.7%) of these yielded positive results for MTB that included samples of: deactivated culture resuspensions exposed to 80 degrees C for 20 minutes, smear preparations and protein extractions. Test procedures were consequently modified and tested again (n = 18), resulting in 0% viability.
  
  
  CONCLUSIONS
  This study demonstrates that it cannot be assumed that conventional practices (i.e. smear preparation) or extraction techniques render the organism non-viable. All methodologies, new and existing, should be examined by individual laboratories to validate the safe removal of material derived from MTB to the outside of a CL3 laboratory. This process is vital to establish in house biosafety-validated practices with the aim of protecting laboratory workers conducting these procedures.
authors.affiliation: National Reference Centre for Mycobacteriology, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada. kym_blackwood@hc-sc.gc.ca
authors.forename: Kym S
authors.initials: KS
authors.lastname: Blackwood
authors.forename: Tamara V
authors.initials: TV
authors.lastname: Burdz
authors.forename: Christine Y
authors.initials: CY
authors.lastname: Turenne
authors.forename: Meenu K
authors.initials: MK
authors.lastname: Sharma
authors.forename: Amin M
authors.initials: AM
authors.lastname: Kabani
authors.forename: Joyce N
authors.initials: JN
authors.lastname: Wolfe
mesh_terms: D019411:Clinical Laboratory Techniques / Q000592:standards*; D003264:Containment of Biohazards / Q000379:methods / Q000592:standards*; D016172:DNA Fingerprinting; D004269:DNA, Bacterial / Q000302:isolation & purification; D050296:Microbial Viability; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification; D016272:Occupational Health; D012150:Polymorphism, Restriction Fragment Length; D018570:Risk Assessment
chemical_list: D004269:DNA, Bacterial