Abstract
BackgroundIn 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.MethodsAn 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.ResultsOf 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°C for 20 minutes, smear preparations and protein extractions. Test procedures were consequently modified and tested again (n = 18), resulting in 0% viability.ConclusionsThis 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.
Highlights
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
In 1987, a 25 year review at the National Animal Disease Center (NADC) described while only 35% of infections at the strict Biological Laboratory at Fort Detrick, MD, had a reportable, documented cause, the NADC could not account for 73% of laboratoryacquired infections (LAIs) occurring at its own facility [2]
All work completed was performed in a CL3 environment, using Class II Type B2 biosafety cabinets (BSCs), and personal protective equipment (PPE) including N100 particulate respirators, double gloves, and protective gowns
Summary
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. In 1976, Robert Pike prepared an extensive summary based on both published reports and surveys of 3921 LAIs that included both M. tuberculosis and other pathogens as the infectious agent [3] He reported that laboratory and mortuary workers that are exposed to tubercle material have a TB incidence rate three times higher than that of the general population and indicated that only 18% of infections could be traced back to a known event. In 1987, a 25 year review at the National Animal Disease Center (NADC) described while only 35% of infections at the strict Biological Laboratory at Fort Detrick, MD, had a reportable, documented cause, the NADC could not account for 73% of LAIs occurring at its own facility [2]. Surveys suggest actual incidence of LAIs with MTB is greater than the amount of reported cases illustrate: these occurrences are likely underestimated due to the nature and length of the disease progression (i.e. workers move or retire before becoming symptomatic), and underreported to the social stigma attached [1,3,5]
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