Laboratory biosafety is the process of applying a combination of administrative controls, containment principles, practices and procedures, safety equipment, emergency preparedness, and facilities to enable laboratory staff to work safely with potentially infectious microorganisms; biosafety also aims at preventing unintentional exposure to pathogens or their accidental release. This manual describes the minimum biosafety measures that should be implemented at the different levels of tuberculosis (TB) testing laboratories to reduce the risk of a laboratory-acquired infection.
The recommendations and approaches in this manual should not replace a country’s existing biosafety guidance when specific requirements already exist for TB laboratories and procedures. Rather, this manual should be used by laboratory directors, managers, biosafety professionals and programmes to inform and guide the implementation of the minimum requirements for individual laboratories and laboratory networks that perform laboratory testing and procedures associated with TB diagnosis.
Risk assessment is an approach that promotes the consideration of risk and the development of appropriate biosafety practices in laboratories based on the unique combination of test procedures, staff expertise and facilities present in each laboratory. While risk assessment is optimally performed at the level of the individual laboratory, this may not be possible, especially in the tens of thousands of peripheral laboratories that perform relatively lower risk procedures in countries that have a high burden of TB and limited resources for local support and oversight. This manual therefore provides pragmatic recommendations for networks of TB laboratories, and focuses on specific procedures, such as microscopy, culture, drug-susceptibility testing (DST) and molecular testing.
Process of developing the biosafety manual
This manual on biosafety for TB laboratories is adapted from the WHO’s Laboratory biosafety manual, 3rd edition.² The contents have been shaped by the outcomes of a technical consultation between the World Health Organization (WHO) and the United States Centers for Disease Control and Prevention (CDC) (September 2008), a WHO Expert Group meeting on biosafety as it relates to diagnostic procedures for TB in the laboratory (April 2009) and the consensus achieved by an independent external review (August 2011).
The manual focuses on addressing the specific needs of TB-control programmes, and facilitating the implementation of effective biosafety measures tailored to multi-tiered TB laboratory systems. At the same time, this manual should be read in conjunction with WHO’s Laboratory biosafety manual since general aspects of laboratory biosafety are covered in that manual, such as the handling of hazardous chemicals not specific to a TB laboratory, fire and other hazards, the transport of infectious substances, and training.
The Expert Group meeting
An Expert Group meeting was convened by WHO in Geneva, Switzerland. Only participants who attended the meeting in person took part in the initial discussion and follow-up discussions where recommendations were made. Individuals were selected to join the Expert Group to represent and balance important perspectives necessary for formulating guidance on laboratory biosafety specifically related to TB. The Expert Group included technical experts, end-users, manufacturers of biological safety cabinets, and biosafety professionals. (Members of the group are listed in Annex 1.)
Declarations of Interest
Members of the Expert Group completed Declarations of Interest. Their responses can be found in Annex 2. These were reviewed by WHO’s legal department prior to the meeting, and the statements were summarized by the chair of the Expert Group at the start of the meeting. Representatives from two companies (Peter van’t Erve and Scott Kreitlein) were declared to have significant conflicts of interest, and were granted observer status; they did not participate in the development of any recommendations in this manual.
External peer review process
An external technical review of this manual was convened at WHO’s Headquarters. Partners’ concerns were addressed where possible and thus informed the manual. A list of people who participated in the peer review process is given in Annex 3.
Rationale and process
The rationale for deviating from previous guidance is explained in the next section. In addition, text boxes with the heading “Expert Group Recommendation” are used to explain where and why current recommendations differ from WHO’s Laboratory biosafety manual.
The process for synthesizing the evidence and developing these guidelines was reviewed and approved by WHO’s Guidelines Review Committee¹ in May 2012.The target date for the next review is 2017.
How this manual differs from WHO’s
Laboratory biosafety manual, 3rd edition
Procedural risk assessment for TB laboratory networks
WHO’s Laboratory biosafety manual² recommends conducting risk assessments for each individual laboratory to identify appropriate practices, approaches and precautions. This manual differs by providing pragmatic recommendations based on laboratory procedures used specifically to diagnose TB and that are typically conducted by different levels of TB services. These recommendations should guide national TB reference laboratories that manage national or regional networks of TB laboratories towards a better understanding of the risks associated with performing certain procedures; the recommendations should also enable national reference laboratories to implement appropriate biosafety practices within suitable facilities, and ensure that adequately trained staff perform a standard range of diagnostic tests for TB.
In many resource-limited high-burden settings, there is insufficient biosafety expertise available to enable national programmes to conduct individualized risk assessments for all laboratories. To assist these programmes, a consultative, consensus-building process was followed to assess the risks usually found in TB laboratories in these settings, and to develop minimum standards to ensure that laboratory testing for TB is conducted safely.
Standards used to develop the guidelines
In 2008, the European Committee for Standardization published laboratory biorisk management standard CWA 15793,³ which highlighted the key factors that need to be considered to successfully establish and implement a biorisk management system. The standard supports the use of a risk-based approach and does not use risk classifications for biological agents or laboratory safety, or the containment levels as described in WHO’s Laboratory biosafety manual. The principles established in CWA 15793 were used to develop this biosafety manual and provide guidance on the minimum requirements for TB facilities conducting diagnostic procedures.
Use of risk-group classifications
The Laboratory biosafety manual recommends that countries draw up national or regional classifications of microorganisms by risk group. A risk-group assignment for a pathogen may vary by geography or by strain because of differences in the epidemiological characteristics of the pathogen in a community or the risk of a laboratory-acquired infection.
It is important to recognize that individuals in a laboratory may differ in their susceptibility to developing TB if they become infected, and that only a small fraction of infected individuals develop active disease over their lifetime.⁴ Individuals with reduced immunity, such as that due to HIV infection or pregnancy, may be at a higher risk of developing TB, and additional precautions may be necessary.
Consequently, and in accordance with standard CWA 15793, this manual takes a risk-based approach that does not use risk classification for biological agents or laboratory safety, or the containment levels as described in the Laboratory biosafety manual.
Designation of biosafety level
The Laboratory biosafety manual describes a four-tier classification system of biosafety. Biosafety levels are based on the composite of the design features, construction, containment facilities, equipment, practices, and operational procedures required for working with agents from various risk groups. It is often erroneously assumed that a microorganism assigned to a particular risk group (for example, Risk Group 3) requires a laboratory of an analogous biosafety level (that is, Biosafety Level 3) for work to be conducted safely. However, a higher level or lower level of biosafety may be more appropriate based on the specific procedure being performed and other factors (see Chapter 1 in this manual).
The Laboratory biosafety manual states that the biosafety level assigned to the specific work being done is driven by professional judgement based on an assessment of the risk rather than by automatic assignment of a laboratory biosafety level according to the particular risk group assigned to a pathogenic agent. The approach developed in this manual builds on the guidance in the Laboratory biosafety manual and uses a procedural approach to risk assessment. TB is predominantly an airborne infection.⁴ Rather than assigning a particular biosafety level to certain procedures, this manual defines the minimum requirements necessary to mitigate risks associated with performing a particular procedure, taking into consideration the risk of aerosolization, the facilities available, and the equipment, practices and procedures required to limit infection.
Mitigating the risks
Using biological safety cabinets
Laboratory-acquired infections often result from the unrecognized production of infectious aerosols containing tubercle bacilli. For laboratories conducting TB testing, the most important hazard (or risk) is the generation of infectious aerosols since infection with Mycobacterium tuberculosis occurs primarily through the inhalation of infectious aerosols, although it can also occur through direct inoculation or ingestion. Infectious aerosols may be generated during the manipulation of liquids containing tubercle bacilli. After settling on surfaces, droplet nuclei are not reaerosolized and are considered noninfectious.⁵ ⁶ ⁷ That is, M. tuberculosis bacteria are usually transmitted only through air, not by surface contact.⁸
Two important considerations in evaluating the risk of aerosolization are the bacillary load of the materials being manipulated and the likelihood of generating infectious aerosols from the material. For sputum specimens (the most common specimen investigated for TB), the bacillary load ranges from 0 (this is the case for up to 90% of diagnostic samples) to 10³ -10⁴/ml in a sputum specimen with a scanty smear grading, to 10⁶/ml in a sample with a 3+ grading.⁹ In a culture grown from sputum specimens, the bacillary load may exceed 10⁸/ ml. Due to the viscosity of sputum specimens, the likelihood of generating an infectious aerosol while manipulating such specimens is much lower than the likelihood of generating an infectious aerosol from a liquid culture. Consequently, the risk associated with manipulating a direct sputum sample is significantly less than that associated with handling cultured material.
This manual differs from WHO’s Laboratory biosafety manual in concluding that biological safety cabinets (BSCs) are not mandatory when performing direct sputum-smear microscopy. The Expert Group recognized that infections with M. tuberculosis are a proven risk to laboratory personnel as well as others who may be exposed to infectious aerosols generated by certain procedures. There remains limited evidence on the risks associated with specific procedures in the TB laboratory. A retrospective study in Korea¹⁰ showed that the relative risk of becoming infected with TB for technicians performing direct acid-fast bacilli (AFB) smear microscopy compared with the general population was 1.4 (95% confidence interval [CI], 0.2–10.0); the risk was 21.5 (95% CI 4.5–102.5) for technicians performing drug-susceptibility testing (DST). The Expert Group concluded that BSCs are not mandatory for performing direct sputum-smear microscopy. The Expert Group found that with good microbiological technique, direct sputum-smear microscopy entails a low risk of generating infectious aerosols, and such procedures may therefore be performed on an open bench, provided that adequate ventilation can be assured. This recommendation is consistent with previous guidance.¹¹ ¹²