Biosafety Level

Biohazard levels, more commonly referred to as “biological safety levels” or “biosafety levels,” are classifications of safety precautions necessary to be applied in the clinical microbiology laboratory depending on specific pathogens handled when performing laboratory procedures. Developed by the Centers for Disease Control and Prevention (CDC), this principle provides a way for medical laboratory scientists and other lab personnel to identify and limit any biological hazards and further reducing the risk in the laboratory. Biohazard levels, on the other hand, also support the principle of biosecurity, which aims at preventing the use of microorganisms as harmful biological agents.

Laboratory associated infections continue to become sources of diseases to the laboratory workforce; this is because of the failure in implementing fundamental control measures in the laboratory which stems from the most effective control which is "elimination", followed by "substitution", "engineering controls", "administrative", and "personal protective equipment" (e.g., respirator use) as the least effective.

Four classifications of biosafety levels (BSLs) exist. Each level contains specific recommendations for a clinical microbiology laboratory with a focus on laboratory practices, safety equipment, and facility construction. As each level progresses, it includes additional biosafety considerations from the previous level. For example, BSL-2 has kept the components of BSL-1 with further requirements, and the same applies to BSL-3 (BSL-2 with additional requirements) and BSL-4 (BSL-3 with additional requirements). The complexity of each level aligns with infectivity, disease severity, the microorganisms’ ability for transmission (including exposure routes), and the nature of the laboratory work to be performed.

Biosafety Levels

1.     Biosafety Level 1 (BSL-1)

BSL-1 labs are used to study infectious agents or toxins not known to consistently cause disease in healthy adults. Microorganism in this level only pose “minimal hazards” to the laboratory and community. They follow basic safety procedures, called Standard Microbiological Practices, and require no special equipment or design features. Standard engineering controls in BSL-1 laboratories include easily cleaned surfaces that can withstand the basic chemicals used in the laboratory.

Biohazard Level 1 usually includes viruses and bacteria like Escherichia coli and chickenpox and many non-infectious bacteria. The level of precaution at this level is minimal and usually involves wearing a face mask and no close contact.

2.     Biosafety Level 2 (BSL-2)

BSL-2 laboratories are used to study moderate-risk infectious agents or toxins that pose a risk if accidentally inhaled, swallowed, or exposed to the skin. Microorganism in this level only pose “moderate hazards” to the laboratory and community. Design requirements for BSL-2 laboratories include hand washing sinks, eye washing stations in case of accidents, and doors that close automatically and lock. BSL-2 labs must also have access to equipment that can decontaminate laboratory waste, including an incinerator, an autoclave, and/or another method, depending on the biological risk assessment.

Biohazard Level 2 usually involves microorganisms that are responsible for mild infections in humans and are often difficult to contract via aerosolized particles, like hepatitis A, B, and C, Lyme disease, Salmonella, measles, mumps, HIV, and dengue. Laboratory personnel can carry out diagnostic tests on the specimens but need to wear gloves, facial protection, and a gown. Additionally, standard precautions at this level should be applied when handling clinical samples from the current outbreak investigations of acute respiratory distress syndrome (ARDS) caused by the Coronavirus Disease 2019 (COVID-19).

3.     Biosafety Level 3 (BSL-3)

BSL-3 laboratories are used to study infectious agents or toxins that may be transmitted through the air and cause potentially lethal infection through inhalation exposure. Microorganism in this level pose “serious hazards” to the laboratory and community. Researchers perform all experiments in biosafety cabinets that use carefully controlled air flow or sealed enclosures to prevent infection. BSL-3 laboratories are designed to be easily decontaminated. These laboratories must use controlled, or “directional,” air flow to ensure that air flows from non-laboratory areas (such as the hallway) into laboratory areas as an additional safety measure.

Other engineered safety features include the use of two self-closing, or interlocked, doors, sealed windows, and wall surfaces, and filtered ventilation systems. BSL-3 labs must also have access to equipment that can decontaminate laboratory waste, including an incinerator, an autoclave, and/or another method, depending on the biological risk assessment.

Biohazard Level 3 includes microorganisms that can be fatal to humans but for which vaccines and other treatments are available. Aside from Mycobacterium tuberculosis, this grouping also includes anthrax, many types of viral encephalitis, hantavirus, Rift valley fever, malaria, Rocky Mountain spotted fever, and yellow fever.

4.     Biosafety Level 4 (BSL-4)

BSL-4 laboratories are used to study infectious agents or toxins that pose a high risk of aerosol-transmitted laboratory infections and life-threatening disease for which no vaccine or therapy is available. The laboratories incorporate all BSL 3 features and occupy safe, isolated zones within a larger building or may be housed in a separate, dedicated building. Access to BSL-4 laboratories is carefully controlled and requires significant training.

Biosafety level 4 (BSL-4) is the highest and "most complex" biohazard level, involving a relatively few clinical microbiology laboratories.

Biohazard Level 4 usually includes dangerous viruses like Ebola, Marburg virus, Lassa fever, Bolivian hemorrhagic fever, and many other hemorrhagic viruses found in the tropics. Only specific persons can work with these viruses, and it requires them to wear a positive pressure personnel suit, with a segregated air supply. There is no treatment available for these viruses, and extreme isolation precautions are mandatory. The CDC has many recommendations on how to manage these viruses. There are no bacteria in this group.

There are two types of BSL-4 laboratories:

a. Cabinet laboratory – all work with infectious agents or toxins is done in a Class III Biosafety Cabinet​ with very carefully designed procedures to contain any potential contamination. In addition, the laboratory space is designed to also prevent contamination of other spaces.

b. Suit laboratory – Laboratory personnel are required to wear full-body, air-supplied suits, which are the most sophisticated type of personal protective equipment​. All personnel shower before exiting the laboratory and go through a series of procedures designed to fully decontaminate them before leaving.

The engineering controls required are different for BSL-4 cabinet and suit laboratories. For either type, they are extensive and supplemented by carefully designed procedures and practices.

RELATION OF RISK GROUPS TO BIOSAFETY LEVELS
Risk Group	Biosafety Level	Laboratory Type	Laboratory Practices	Safety Equipment
1	Basic – Biosafety Level 1	Basic Teaching, Research	GMT	None; Open Bench Work
2	Basic – Biosafety Level 2	Primary Health Services; Diagnostic Services, Research	GMT plus protective clothing, Biohazard Sign	Open bench plus BSC for potential Aerosols
3	Containment – Biosafety Level 3	Special Diagnostic Services, Research	Same as Level 2 plus special clothing, controlled access, directional airflow	BSC and/or other primary devices for all activities
4	Maximum containment – Biosafety Level 4	Dangerous pathogen units	Same as Level 3 plus airlock entry, shower exit, special waste disposal	Class III BSC, or positive pressure suits in conjunction with Class II BSCs, double–ended autoclave (through the wall) filtered air
BSC – Biologic Safety Cabinet GMT – Good Microbiological Techniques

SUMMARY OF BIOSAFETY REQUIREMENTS
	1	2	3	4
Isolation of Laboratory	NO	NO	YES	YES
Room sealable for decontamination	NO	NO	YES	YES
Ventilation
Inward Airflow	NO	Desirable	YES	YES
Controlled ventilation system	NO	Desirable	YES	YES
HEPA–filtered air exhaust	NO	NO	YES/NO	YES
Double–door entry	NO	NO	YES	YES
Airlock	NO	NO	NO	YES
Airlock with shower	NO	NO	NO	YES
Anteroom	NO	NO	YES	–
Anteroom with shower	NO	NO	YES/NO	NO
Effluent Treatment	NO	NO	YES/NO	YES
Autoclave
On site	NO	Desirable	YES	YES
In Laboratory Room	NO	NO	Desirable	YES
Double–ended	NO	NO	Desirable	YES
Biologic Safety Cabinets	NO	Desirable	YES	YES
Personnel safety Monitoring Capability	NO	NO	Desirable	YES

Risk Categories of Microorganisms

In both the NIH guidelines and the WHO manual, four categories of microorganisms used in laboratory work are recognized. The basis of the classification is the risk of infection to laboratory workers and, in the event of escape from the laboratory to the community. Assigning a microorganism to a risk category is dependent on an initial risk assessment made by the investigator and is based on current knowledge of the:

1. Pathogenicity of the organism (all microorganisms do not cause diseases),

2. Host range and mode of transmission of the organism,

3. Local availability of effective measures to prevent a disease outbreak, and

4. Local availability of effective treatment.

The Appendix B of the NIH guidelines, Classification of Human Etiological Agents based on Hazard, as does Table 1 of the WHO manual, recognizes four Risk Groups of microorganisms:

Risk Group 1: Microorganisms unlikely to cause human or animal diseases and thus pose little or no risk to individuals and to the community; sometimes designated as Generally Regarded As Safe (GRAS) organisms (e.g., asporogenic Bacillus subtilis or Bacillus licheniformis, the K-12 strain of Escherichia coli)

Risk Group 2: Microorganisms that are pathogenic, but unlikely to pose a serious hazard to laboratory workers, livestock, the community, or the environment as effective treatment and preventive measures to limit spread of infection are available. These organisms thus are of moderate risk to the individual and low risk to the community (e.g., Bacterial agents—Aeromonas hydrophila, Escherichia coli, Klebsiella spp., Salmonella spp.; Fungal agents—Penicillium marneffei, Blastomyces dermatitidis; Parasitic agents—Ascaris spp., Trypanosoma spp.; Viruses—Adenoviruses, Coronaviruses, Papilloma viruses)

Risk Group 3: Microorganisms that are pathogenic and can cause serious human or animal diseases but are not contagious or have effective treatment and preventive measures. These organisms pose high risk to the individual, but low risk to the community (e.g., Bacterial agents—Brucella spp., Francisella tularensis, Rickettsia spp.; Fungal agents—Coccidiodes immitis, Histoplasma spp.; Viruses and prions—Togaviruses, Flaviviruses such as the Japanese encephalitis virus, West Nile virus, Pox viruses, prions such as the transmissible spongiform encephalopathies, retroviruses such as Human Immunodeficiency Virus, rhabdovirus)

Risk Group 4: Microorganisms that usually cause serious diseases in humans and animals and can be readily transmitted either directly or indirectly from one to the next individual. Effective treatment and preventive measures are usually unavailable. This class of organisms thus poses a high risk to individuals and to the community (e.g., Viral Agents such as the Lassa virus, Ebola virus, Marburg virus, Herpes virus simiae, Kayasanur Forest disease, Central European encephalitis, and yet unidentified hemorrhagic fever agents).

The NIH guidelines recognize that this classification is dependent on current knowledge of pathogenicity, and with the development of better therapeutic and preventive measures, pathogens may be assigned to a lower risk category. Different countries may assign the same organism to different risk groups, possibly because the same organism is more virulent in certain parts of the world than others depending on climatic conditions and other factors. Also, any strain more virulent than the wild-type parent strain should be assigned to a higher risk group.

Good Laboratory Practice (GLP)

Good Laboratory Practice embodies a set of principles that provide a framework within which laboratory studies are planned, performed, monitored, recorded, reported, and archived.

The primary purpose of GLP is to ensure uniformity, consistency, and reliability of safety tests (nonclinical) for pharmaceuticals, agrochemicals, aroma and color food/feed additives, cosmetics, detergents, novel foods, nutritional supplements for livestock, and other chemicals. These safety tests are used to generate data on various parameters from physicochemical properties to toxicity (nonclinical) for use of regulatory authorities to make risk/safety assessments.

Originally, GLP regulations were intended for toxicity testing only and were reserved for laboratories undertaking animal studies for preclinical work. GLP is now followed in all laboratories where research or marketing studies are to be submitted to regulatory authorities such as the FDA. Establishment of GLP is mandatory to evaluate safety or toxicity of products intended to undergo clinical trials.

Compliance with GLP requires that:

1. The tests should be conducted by qualified personnel.

2. Each study should have a Study Director responsible for the overall conduct of the tests.

3. The laboratory study and the accompanying data should be audited by a Quality Assurance Unit.

4. All laboratory activities must be performed in accordance with written and filed management-approved Standard Operating Procedures (SOPs). SOPs should cover policies, administration, equipment operation, technical operation, and analytical methods.

5. All control and test articles and reagents must be identified, characterized, and labelled with information regarding source, purity, stability, concentration, storage conditions, and expiration date.

6. The equipment must be maintained, calibrated, and must be designed to meet analytical requirements.

Compliance with GLP has served to harmonize test methods across nations, facilitating generation of mutually acceptable data, thus avoiding duplication of tests, and saving time and resources.