Biosafety Recommendations for the Contained Use of M. tuberculosis

The WHO and the CDC classify M. tuberculosis among the pathogens that require a biosafety level 3. Based on the risk assessment and according to technical characteristics, safety equipment and work practices (Philippe et al., 2006).
The following recommendations for the contained use of M. tuberculosis are proposed (CDC, 1999; WHO, Laboratory Safety Manual, 2004):
1) Any containers used for collecting clinical specimens could be contaminated with tubercle bacilli, primary or secondary culture samples or any other material known to contain M. tuberculosis should be opened in a class I or II biosafety cabinet (BSC). Personnel wearing gloves should disinfect the outside of the container.
2) For the laboratory involved in the diagnosis of tuberculosis, direct smear examination and primary culture of specimens require to work in BSL-2 facilities. Primary cultures only concern cultures obtained directly from clinical specimens in solid or liquid culture medium. They should be performed in "unbreakable" vials. These laboratories should send the positive primary culture, without any subsequent examination, to a BSL-3 laboratory for further analysis.
3) When the diagnostic or research laboratory is involved into subsequent characterization of the tubercle bacilli by means of secondary cultures, antimicrobial susceptibility testing, and any other test performed on primary or secondary living cultures, BSL-3 facilities, equipment and work practices should be used.
4) Needle sampling through vial's septum (e.g. for smear examination, nucleic acid amplification or any other biological test) should not be performed.
5) Contaminated pipettes should be discarded horizontally in a container immediately after use. This container must be dry in order to avoid aerosol production pipette laying down.
6) Disposable plastic bacteriological loops are preferable; if wire loops are used, they must be sterilized in an electrically operated “micro-incinerator”. Alternatively, they may be submerged into a flask filled with sand and 90% alcohol, before they are flamed.
7) Needles and syringes or other sharps should be restricted in the laboratory and only used when there is no alternative: only disposable syringe-needle units (i.e., needle are sealed to the syringe) should be used for injection or aspiration of infectious material.
8) Contaminated syringes should be carefully discarded after use in special puncture-resistant containers used for sharps disposal.
9) Appropriate systems of respiratory protection with HEPA filtration should be worn when aerosols cannot be safely contained or for the handling of positive cultures in the BSC.
10) The slides used for AFB smear identification should be handled with care to prevent contamination of hands and discarded after use as potentially contaminated waste.
Smears, which may contain M. tuberculosis, should be stored in a closed box as it was shown that viable tubercle bacilli could be excreted by cockroaches following ingestion from heat-fixed smears (Allen, 1987).
Flow cytometry applications involving M. tuberculosis should take into account recent publications and specific biosafety guidelines (Schmid et al., 2003).
Disinfection, inactivation of M. tuberculosis and waste management
The high lipid content of the cell wall confers to the mycobacteria a great resistance to classical disinfectants. The bacilli are generally more resistant to chemical disinfection than other vegetative bacteria. Their resistance to disinfectants is considered intermediate between other non-sporulating bacteria and spores (Kunz & Gundermann, 1982).
The acquired multidrug resistance does not seem to modify the resistance to disinfectants (Sattar et al., 1995). Quaternary ammoniums inhibit tubercle bacilli but do not kill them. M. tuberculosis is also resistant to acids and alkali. Mercurial compounds are considered to be ineffective against the mycobacteria. Efficient disinfectants are 5% phenol, 5% formaldehyde during at least ten minutes, 2% glutaraldehyde during 30 minutes exposure or sodium hypochlorite (5%) during one minute. Ethyl and isopropyl alcohols in high concentrations are generally accepted to be excellent mycobactericidal agents. 70% ethyl alcohol can be used as surface disinfectant. Formaldehyde vapours can be used to disinfect BSC's and facilities. Iodine and ionophores are considered to be effective against mycobacteria and are generally used in combination with ethyl alcohol (Rubin, 1991).
It is recommended to test killing methods used on M. tuberculosis suspension before removal from Biosafety Level 3 laboratory. A study compared the efficacy of several disinfectant mixtures on class of risk 3 M. tuberculosis Erdman strain. It was observed that fixatives containing low concentration of glutaraldehyde alone are not efficient to kill M. tuberculosis. The use of a combination of 2% paraformaldehyde and 2% glutaraldehyde or a solution of 5% formalin is recommended for M. tuberculosis inactivation (Schwebach et al., 2001). Another experimental study has demonstrated that all tubercle bacilli killing methods should be validated by individual laboratories before removing material derived from M. tuberculosis to the outside of the BSL-3 laboratory (Blackwood et al.,2005).
Work surfaces should be decontaminated at least once a day with an appropriate disinfectant and immediately after any accidental contamination with infectious materials. Laboratory workers should disinfect their hands after manipulations with an appropriate disinfectant, after removing gloves, and before leaving the laboratory. Worn gloves and protecting clothes should be autoclaved before leaving the laboratory (Philippe et al., 2006).
Attention should be given to waste inactivation. Decontamination by autoclaving or incineration is essential. Ideally, an autoclave for the sterilisation of contaminated materials should be available in or adjacent to the laboratory. If the inactivation takes place outside the laboratory (autoclave or incinerator), wastes should be placed in a leak proof bag or an unbreakable and leak proof container (for liquid wastes), sealed and disinfected on the outside before removal from the laboratory. In addition to the international Biohazard symbol, bags or containers should be adequately labelled to prevent opening before decontamination. Removal of bags and containers should be performed according to written procedures. (Kimman et al., 2008b).
Generally, special care should also be taken for the following manipulations (Philippe et al., 2006):
1) Acid-fast staining (AFB smear): smear fixation on slides (by heat or methanol) can generate aerosols. Although fixed smear may still contain viable organisms, they are not easily aerosolized.
2) Manipulation of solid and liquid cultures: Unlike sporulating fungi or bacteria, the opening of a Petri dish or a tube lid containing mycobacteria is not thought to pose a real risk. However, manipulation of the colony mass increases the likelihood of dispersal of the tubercle bacilli into the air, especially when organisms are incinerated from the bacteriological needle or loop. In case of accidental breakage involving culture tubes, a culture of M. tuberculosis grown on a solid medium is rated as producing a “minimal” aerosol requiring local disinfection.
3) flow cytometry: applications of flow cytometry in clinical microbiology and research laboratories are numerous with direct detection of infected cells or isolated mycobacteria, serological tests, monitoring of infections, antimicrobial therapies and cell-sorting
4) In experimental settings, flow cytometry has also been used to assess sputum decontamination methods improvement. Flow cytometry analysis and/or sorting procedures can generate aerosols containing viable M.tuberculosis