title: Use of RODAC plates to measure containment of Mycobacterium tuberculosis in a Class IIB biosafety cabinet during routine operations.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Mycobacteriology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium. Electronic address: gdaneau@itg.be.
authors.forename: Géraldine
authors.initials: G
authors.lastname: Daneau
authors.affiliation: Mycobacteriology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.
authors.forename: Elie
authors.initials: E
authors.lastname: Nduwamahoro
authors.affiliation: Mycobacteriology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.
authors.forename: Kristina
authors.initials: K
authors.lastname: Fissette
authors.affiliation: Perseus BVBA, Technologiepark-Zwijnaarde, Zwijnaarde, Belgium.
authors.forename: Patrick
authors.initials: P
authors.lastname: Rüdelsheim
authors.affiliation: Mycobacteria Reference Laboratory, National Institute for Public Health and the Environment, Bilthoven, The Netherlands.
authors.forename: Dick
authors.initials: D
authors.lastname: van Soolingen
authors.affiliation: Mycobacteriology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium; Division of Infectious Diseases, New York University, New York, NY, USA; Vaccination Theme, Medical Research Council Unit, Fajara, The Gambia.
authors.forename: Bouke C
authors.initials: BC
authors.lastname: de Jong
authors.affiliation: Mycobacteriology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium; Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium.
authors.forename: Leen
authors.initials: L
authors.lastname: Rigouts
mesh_terms: D001431:Bacteriological Techniques / Q000295:instrumentation / Q000379:methods*; D003264:Containment of Biohazards / Q000295:instrumentation*; D004866:Equipment Contamination; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification
keywords: Biosafety; Environmental sampling; Ethanol; Propidium monoazide; Replicate Organism Detection and Counting; Tuberculosis