ABSTRACT
Objective/background: guidelines for the manipulation of Mycobacterium tuberculosis [MTB] cultures require a Biosafety Level 3 [BSL-3] infrastructure and accompanying code of conduct. In this study, we aimed to validate and apply detection methods for viable mycobacteria from surfaces in a BSL-3 MTB laboratory
Methods: we evaluated phenotypic [Replicate Organism Detection and Counting [RODAC] plates] and molecular [propidium monoazide [PMA]-based polymerase chain reaction [PCR]] approaches for the detection of viable mycobacteria, as well as the effect of 70% ethanol applied for 5 min for disinfection against mycobacteria. For validation of the method, recovery of serial dilutions of Mycobacterium bovis bacillus Calmette-Guerin from glass slides was measured. Subsequently, we stamped surfaces in and around the biosafety cabinet [BSC] after different technicians had manipulated high bacterial load suspensions for routine drug-susceptibility testing in a Class II BSC
Results: RODAC stamping could detect as few as three bacteria on slides stamped either 5 min or 60 min after inoculation. PMA-based PCR, tested in parallel, did not pass validation. Mycobacteria were still detected after 5-min disinfection with ethanol 70%. In the BSL-3, from 201 RODAC-stamped surfaces, MTB was detected in four: three inside a BSC- on a tube cap and on an operator's gloves-and one outside, on an operator's gown
Conclusion: RODAC plates detect mycobacteria at low numbers of microorganisms. In addition, this method allowed us to show that 70% ethanol does not reliably kill mycobacteria when applied for 5 min to a dried surface, and that MTB bacilli may arrive outside a Class II BSC during routine practice, although the route could not be documented