4Moderate-risk TB laboratories

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The recommendations in this chapter are the minimum requirements needed to limit or reduce risks of infection in laboratories carrying out specific procedures that are considered to have a moderate risk of spreading TB. Additional measures may be deemed necessary following a site-specific risk assessment.

Moderate-risk laboratories that follow the minimum biosafety requirements described in this chapter can safely perform certain procedures that entail a moderate risk of specimen aerosolization with a relatively low concentration of infectious particles. Moderate-risk laboratories can:

  • process specimens for inoculation on primary solid-culture media;
  • perform direct DST (for example, direct line-probe assays, Microscopic observation drug susceptibility [MODS], Nitrate reductase assay [NRA] on processed sputum).

4.1. Factors that increase the risk of infection

In addition to the general risks that are addressed by the biosafety measures described in Chapter 2 (such as unauthorized persons in the laboratory, mouth pipetting, cluttered work stations, improper waste disposal), the TB laboratory classified as moderate risk also faces the following challenges, all of which increase risks:

  • staff may work in areas with poor ventilation;
  • they may work with poor illumination;
  • BSCs may be poorly maintained and not certified;
  • BSCs may not be properly ducted;
  • the work environment may be dusty, and high-efficiency particulate air (HEPA) filters in BSCs may become blocked;
  • careless manipulation of specimens may lead to aerosolization;
  • precautions for using the vortex may not be followed properly (for example, it may be used outside the BSC);
  • specimen containers may break or leak during centrifuging;
  • problems may be associated with opening centrifuge buckets outside the BSC;
  • adequate warnings of biohazards may be lacking, and information on who should be contacted during an emergency may be inadequate;
  • cooling or heating systems may not work properly.

Good microbiological techniques are essential to minimize the risk of aerosolization.

4.2. Specific features and essential minimum biosafety measures

In a laboratory where there is a moderate risk of infection, there are two levels of containment: the BSC (primary containment) and the laboratory itself (secondary containment). To address the specific risks associated with a moderate-risk laboratory, the following mitigation and control measures should be established.


Biological safety cabinets: All processing and digestion of sputum samples and manipulation of liquefied sputum specimens must be conducted in a BSC. The cabinet is the primary form of containment while specimens are processed for culture inoculation or for performing direct DST. Hence, good microbiological techniques and proper use of the cabinet are critical to allow work to be conducted safely.

Improper use of the cabinet allows aerosols to be released into the laboratory. (See Chapter 6 for more information on BSCs.)

BSCs should be sited away from thoroughfares and out of cross-currents from doorways and air-inlet systems. Air expelled from properly maintained cabinets will have passed through HEPA filters at the top of the cabinet and so can be expelled either into the room or ducted to the outside, depending on the degree of sophistication of the ventilation system installed.

Adequate space is needed between the cabinet and the ceiling to ensure that the air flowing from the cabinet is not impeded.

Class I or Class II cabinets are recommended; however they must be designed by a certified manufacturer and regularly maintained. They must be certified to be functioning properly on site at least annually. Class II type A2 cabinets are preferable because they offer protection both for personnel and the media being inoculated (product protection).

Class II type B cabinets are suitable but are not recommended for new TB laboratories because they require hard-ducting. Additionally, it is more difficult to balance and maintain them, and ensure they are functioning properly. Hard-ducting requires that the building's exhaust system be precisely matched to the airflow requirements of the manufacturer.

An uninterrupted power supply for the cabinet and the exhaust fan is necessary in settings where power is unreliable; this supply gives laboratory staff time to safely complete any hazardous work, and for contaminated air remaining in the cabinet to be vented outside. Devices to prevent the backflow of air should be installed in the ducting of cabinets to stop potentially contaminated air from flowing back into the laboratory in the event of a power failure.

It is helpful to have a stand-by generator for the cabinet and other essential equipment, such as incubators and freezers.


Ventilation: In addition to the BSC (the primary barrier), the secondary barrier (provided by the laboratory itself) is achieved by maintaining a unidirectional airflow into the laboratory, and by ensuring there are a minimum of 6–12 ACHs.

A simple means of creating unidirectional airflow is to place a vent that allows air to flow into the clean area of the laboratory and to operate continuously one or more thimble-fitted BSCs to draw air towards the dirty area, remove the air from the laboratory, and expel it outside the building. A visual monitoring device with or without an alarm should be installed so that staff can ensure at all times that proper directional airflow is maintained in the laboratory (see Box 5).

Ducting the BSC to the outside using a thimble connection helps create unidirectional airflow into the laboratory, and any contaminated air in the BSC is expelled from the laboratory through the HEPA filters in the BSC. When the cabinet is turned on, the external fan extracts air from both the cabinet and the room. When the cabinet is switched off, the expelled air will be extracted only from the room. An external fan can be installed with or without a link to the status (operating or stand-by) of the cabinet. It is best if the external fan has a separate switch from the BSC, or alternatively it can be coupled with a relay circuit so that the external fan continues operating for a given time after the BSC has been turned off to ensure that all of the air expelled from the BSC is vented outside. The major advantage of a thimble connected BSC is that no adjustments need to be made to the cabinet and the direction of air flowing from the laboratory to the outside will be maintained.

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Box 5

How to calculate the number of air changes per hour (ACH) in a laboratory that uses a biological safety cabinet (BSC) ducted with a thimble connection. Determine the volume of the room in the laboratory (floor area × room height). Determine the (more...)

Alternatively, air expelled through the HEPA filters within the BSC can be released into the laboratory. However, in such cases, there must be a separate exhaust system for the building that ensures a minimum of 6–12 ACH in the laboratory. The building's ventilation system must be constructed in such a way that air from the moderate-risk laboratory is not recirculated to other areas within the building.

When air expelled from the laboratory is discharged to the outside of the building, it must be dispersed away from occupied buildings and air intakes.

Windows must be kept closed at all times in moderate-risk and high-risk TB laboratories.

  • Personal protective equipment: Each laboratory must evaluate its risks (for example, by assessing the laboratory's activities and workload, the prevalence of TB and the prevalence of drug-resistant strains) and decide on the level of personal protection that is appropriate for staff. Protective laboratory gowns and gloves must be worn at all times in laboratories where there is a moderate risk of infection.
    During specimen processing, samples are liquefied; this makes it more likely that aerosols will be generated so measures to minimize the production of aerosols are essential.
    Gloves should be changed regularly. Staff must always wash their hands before leaving the laboratory.
    Respirators are not required, provided that specimens are processed within a properly maintained BSC using good microbiological techniques. Respirators should not be seen as an alternative to a BSC.
  • Laboratory design: The laboratory must be separate from the areas that are open to unrestricted traffic flow within the building. A station for hand washing should be provided near the laboratory's exit.
  • Decontamination and waste disposal: All infectious waste must be removed from moderate-risk laboratories for proper disposal. Waste must be transported in sealed plastic bags or containers, following appropriate local regulations. Any materials that are reused must be decontaminated with a suitable disinfectant or autoclaved before being removed from the laboratory.
  • Minimizing the generation of aerosols: Staff training should always include information on the safest methods to use for culture procedures to prevent inhalation of aerosols generated when using loops, pipetting, opening specimen containers, handling damaged or leaking containers, centrifuging and vortexing. The possibility of infectious material spattering when an open Bunsen burner flame is used should be avoided by using an enclosed electric microincinerator to sterilize reusable loops. The use of sterile disposable transfer loops and transfer pipettes is recommended.
    Centrifuges require safety buckets or containment rotors. Infectious materials may be centrifuged in the open laboratory provided that sealed centrifuge safety cups are used and buckets are loaded and unloaded within a BSC.