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.
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.
Blackwood
Kym S
KS
National Reference Centre for Mycobacteriology, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada. kym_blackwood@hc-sc.gc.ca
Burdz
Tamara V
TV
Turenne
Christine Y
CY
Sharma
Meenu K
MK
Kabani
Amin M
AM
Wolfe
Joyce N
JN
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
D004269:DNA, Bacterial