1. Biosafety Recommendations for Laboratory Testing for TB
Thomas M. Shinnick, Ph.D.
Associate Director for Global Laboratory Activities, Division of TB Elimination
Anticipating Biosecurity Challenges of the Global Expansion of High Containment Biological Laboratories
July, 2011
National Center for HIV/AIDS, Viral Hepatitis, STD & TB Prevention
Division of Tuberculosis Elimination

2. Why is Biosafety Needed in the TB Laboratory?
Risk of infection with M. tuberculosis is 3x to 9x higher for TB lab workers than for other lab workers
Infection often results from unrecognized production of infectious aerosols containing tubercle bacilli
Infection can occur from needle sticks, through broken skin, etc.

3. Biosafety Level (BSL)
Conditions under which an infectious agent can ordinarily be safely handled.
Conditions are a combination of:
laboratory practices and techniques
safety equipment
laboratory facilities
Recommended BSLs for many of the infectious agents have been developed
But different methods for the same agent may require different BSLs
So, how does one determine the appropriate BL to use?

4. GLI Biosafety Guidance
Biosafety guidance for TB lab procedures
technical consultations, expert meetings
GLI, WHO, CDC were the lead agencies
Consensus recommendations for minimum biosafety requirements for
direct AFB-smear microscopy
processing specimens to concentrate bacilli for smear, culture, molecular tests
manipulating cultures for smear, subculture, ID, DST, molecular tests

5. Risk Assessments for TB Procedures
Based on likelihood of producing infectious aerosols and the concentration of bacilli
Limited risk for infectious aerosols
direct AFB smear microscopy
Moderate risk for infectious aerosols
processing sputum specimens
High risk for infectious aerosols
processing cultures and suspensions

6. Direct AFB Smear Microscopy
Limited risk of generating infectious aerosols
Work can be done on an open bench
restricted access to the laboratory
separate bench for smear preparation
Adequately ventilated laboratory
6–12 ACH, directional airflow
natural or mechanical ventilation
Proper disposal of infectious material
Primary containment or barriers to protect the worker include

7. Processing Sputum Specimens for Smear, Culture, Molecular Tests – 1
Moderate risk of generating infectious aerosols during specimen manipulation
Laboratories must have restricted access and be separated from public areas
Impermeable surfaces for easy cleaning
Air flows into lab without re-circulation to non-lab areas (directional airflow)
6–12 ACH, closed windows
Proper disposal of infectious material

8. Processing Sputum Specimens for Smear, Culture, Molecular Tests – 2
Class I or II Biosafety Cabinets used for all open manipulation of agents
BSCs must be properly installed and certified at least annually
BSC exhaust may be
ducted to outside using a hard duct or thimble fitting (preferred)
recirculated into the room if assured that the BSC is functioning properly
Use aerosol-containment rotors
Primary containment or barriers to protect the worker include

9. Processing Cultures for Smear, ID, Subculture, DST, Molecular Tests – 1
High risk of generating infectious aerosols during manipulation of liquid suspensions
Work done in a containment lab which has restricted access and a double door entry
Impermeable surfaces for easy cleaning
sealing room for fumigation is not required
Air flows into lab without re-circulation to non-lab areas (directional airflow)
6–12 ACH, mechanical ventilation, sealed windows
Autoclave available on site
This sort of risk assessment has been done for routine work with a number of infectious agents, and 4 generic biosafety levels developed.

10. BSCs must be properly installed and certified at least annually
Processing Cultures for Smear, ID, Subculture, DST, Molecular Tests – 2
Class I or II Biosafety Cabinet used for all open manipulation of agents
BSCs must be properly installed and certified at least annually
BSC exhaust may be
ducted to outside using a hard duct or thimble fitting (preferred)
recirculated into the room if assured that the BSC is functioning properly
Use aerosol-containment rotors
Primary containment or barriers to protect the worker include

11. TB Laboratory Biosafety Gaps
What are minimum facility requirements?
U.S./European-style BSL3?
containment room, airflow, BSC?
What are suitable laboratory layouts?
What are minimum safety requirements
for technicians who are HIV+?
for areas with high rates of MDR/XDR TB?
When should respirators be required?
Are Class I BSCs adequate?
How to ensure functioning BSCs?

12. BSL3 – Secondary Containment
BSL2 secondary containment plus:
Directional inward airflow without re-circulation to non-lab areas; 6-12 ACH
Controlled access; separate area
Double door entry (airlock)
Enclosures for aerosol-generating equipment
Walls, floors and ceilings are water resistant for easy cleaning
Room penetrations sealed
Secondary containment or barriers to protect the environment include

14. Interim Guidance for XDR TB
Clinical specimens from known or highly suspected XDR TB patients
BSL2 with full BSL3 practices are highly recommended
Manipulation of cultures of XDR TB strains
BSL3 practices, containment equipment, and facilities are required. BSL3 practices must include the use of respiratory protection and the implementation of specific procedures and use of specialized equipment to prevent and contain aerosols.

15. Guidance for Samples from Known or Highly Suspected XDR TB Patients
Manipulation of clinical specimens
‘Moderate risk’ facilities with ‘high risk’ practices & PPE are highly recommended
Manipulation of cultures
‘High risk’ practices, containment equipment, and facilities are required. Practices must include the use of respiratory protection and the implementation of specific procedures and use of specialized equipment to prevent and contain aerosols.

16. Acknowledgements Véronique Vincent CN Paramasivan Chris Gilpin
Daniela Cirillo
Jean Joly
Jenny Allen
John Ridderhof
Jon Crane`
Knut Feldmann
Moses Joloba
Paul Jensen
Peter van't Erve
Philippe Dubois
Sang Jae Kim
Shanna Nesby
Thomas Shinnick
Andrew Ramsay
Karin Weyer
May Chu
Nicoletta Previsani
Sebastien Cognat

17. Thank You
National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention
Division of TB Elimination