Use of RODAC plates to measure containment of Mycobacterium tuberculosis in a Class IIB biosafety cabinet during routine operations.

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 5min for disinfection against mycobacteria. For validation of the method, recovery of serial dilutions of Mycobacterium bovis bacillus Calmette-Guérin 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 5min or 60min 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 5min 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.