Secondary barriers - Facility Design and Construction
Antonello Fadda

Design and engineering features of biosafety laboratories are an highly specialized and involved matter, where different and sometimes conflicting aspects, as safety, security, environmental protection, ergonomy and community concerns need to be addressed using state of the art technology. Moreover this effort is often made in face of budget restrictions and demanding regulatory procedures, which have the effect of prolonging the design and construction phase, sometimes making obsolete the initial specifications and starting an endless cycle of design revisions.
The purpose of this presentation is not to discuss engineering solutions in detail but to provide some basic tools needed to use and manage these facilities with the highest level of safety and efficiency.
In this view the aspect of secondary containment comes out as a central one, being needed at the higher levels of biosafety (BSL3 and BSL4) and being characterized by technical aspects that usually are not fully understood. The rationale of use of this containment method is explained, with a mention to the different technical issues found at the perimeter of the containment area. The management of air is the most challenging one, and a specialized ventilation system is needed to cope with it. The key concept of “negative differential pressure”, also referred as “inward direction airflow” is explained. The general principles and problems of this approach are shown, with the help of an example system. The presentation then discuss in greater detail the points of special concern to the staff, relative to the HVAC system operation. Eventually the aspect of test and controls is given due attention, with guidelines to face the task of certification of secondary barriers. Final remarks include the advocacy of a better communication and cooperation between engineers and laboratory staff and managers. To this purpose the involvement of a skilled commissioning agent may be of great value to facilitate communication between professionals with a different background.

Laboratory Biosafety Equipment
Martien Broekhuijsen

Biosafety equipment is any type of equipment that is used for containing biohazardous materials inside the biosafety area, for preventing the spread of such materials, or for protecting workers against contact with such materials.
The equipment typically used in laboratories has many shapes and sizes and different levels of complexity. Perhaps the most central piece of equipment is the biosafety cabinet, sometimes called the microbiologist’s fumehood. However, this is an unjust description of the design of biosafety cabinets as they do much more than a fumehood. Besides protecting the worker and preventing spread of materials in the laboratory (or outside), they also protect the biological materials from becoming contaminated with biological materials from the environment or even from within the work space inside the cabinet (cross contamination).
Several types of biosafety cabinets exist, with different properties and adapted to different kinds of work or risk level. The proper use of biosafety cabinets is crucial to their performance and workers need to be trained for doing so. The placement of biosafety cabinets in a room and connection to the ventilation system are also important factors to address.
Other examples of laboratory equipment that plays a role in containment or protection are centrifuges, pipettes, refrigerators, freezers, and incubators. Not all standard laboratory equipment is suitable for work with biohazardous materials.
Positive pressure biosafety suits with breathing air supply as used in some BSL4 laboratories are also considered as biosafety equipment. Personal protective equipment can also be regarded as biosafety equipment but these are discussed in a separate lecture.

Personal Protective Equipment
Martien Broekhuijsen

Personal protective equipment (PPE) consists of a wide range of equipment and materials that are worn by a person in order to protect him/her against biohazardous materials. PPE varies from basic, simple materials to complex equipment, but is always individually used (worn) by a person.
Examples are gloves, boots, coveralls, eye and face protection, and breathing protection. The proper use of PPE is essential for biosafety. The user must therefore be trained sufficiently. Training with live agents is very effective for building confidence. Training must include the correct donning (putting on) and doffing (taking off) of PPE. Correct donning is important for obtaining the necessary protection level. Correct doffing is essential for not getting infected afterwards or spreading a biological agent beyond the hotzone.
Decontamination of a person still wearing PPE is often a standard part of work before doffing. Usually a sequence of steps is chosen to exit from the hotzone via the decontamination area to the safe zone. Doffing of PPE in the correct order is part of such a sequence. Some PPE components are disposable (e.g. latex gloves), other parts are too expensive to dispose off. Those parts should be made of materials that are resistant against the decontaminant in use. The material and construction of PPE should enable easy decontamination and cleaning.
There is no standard PPE, not even for a specific biosafety level. PPE must always be adapted to the specific work conditions and surrounding, and especially to the particular infectious organism being handled. Knowledge of the usual routes of infection of an organism helps in selecting the appropriate PPE components.
PPE should not inhibit the work to be carried out. Although safety comes first, sometimes the PPE is slightly adapted to better accommodate specific actions during work. PPE should also not pose a high burden upon the worker, as this might encourage a worker to not consistently using it. This relates to weight and general comfort, but also to heat and moisture building up inside a coverall. Breathing should be easy even under moderately stressful work conditions.

Good Laboratory Practices
Martien Broekhuijsen

Good laboratory practices are all those methods and procedures that in combination with other measures (e.g. physical barriers or equipment) ensure the safe handling of hazardous biomaterials. In several countries the most often used description is Good Microbiological Practice (GMP), not to be confused with Good Manufacturing Practice. Other terms used include Standard Microbiological Practice (SMP) or Safe Microbiological Practice (also SMP).
Any biosafety procedure or biosafety equipment can be rendered useless if the user makes mistakes or does not use the equipment properly. The correct use of equipment and proper execution of methods by the microbiological worker is at the core of biosafety. A proper level of education and sufficient training are the most essential starting points for GMP.
Many international guidelines include paragraphs on GMP, such as from the WHO[1], CDC[2], and others. International biosafety associations such as ABSA and EBSA also publish guidelines for GMP.
GMP involves aspects such as training, peer control, procedures, SOP’s, and other elements. Several organisations offer training courses for GMP. Many use videos and photos to illustrate essential elements of GMP. GMP is not a replacement for a microbiological education, it is merely an extension, relying on a solid microbiological education as basis.

[1] http://www.who.int/csr/resources/publications/biosafety/WHO_CDS_CSR_LYO_2004_11/en/
[2] http://www.cdc.gov/biosafety/publications/bmbl5/

Laboratory Accidents
Davide Mileto

Safety and security of work place are among the most important aspects of the working activity. The risk assessment for the scientific laboratory, chemical and biological, is linked with the presence of material, procedures and hazardous factors in the daily workers’ activities. Among the different elements to be considered in a risk assessment plan there are surely the agents, the different instruments, the problem linked with the space limitation, aspects of organizational-management, sometimes the lack of information, and education and training of the staff. The combination of these factors in a laboratory setting could be to cause of the laboratory accidents: break of tubes in a centrifuge, projection of liquid in the eye, spill of sample, accidental injection of a contaminated solution, the aerosol of liquid solution that could generate a spread of agents into the laboratory environment, which might be the cause of workers infection or contamination of various equipment and materials, etc. A relevant problem in the context of laboratory accidents are all infections acquired through laboratory or laboratory-related activities regardless whether they are symptomatic or asymptomatic in nature. This infections are defined as Laboratory Acquired Infection (LAI) or laboratory-associated infections. Among the LAI, bloodborne Pathogens have a relevant importance especially for the HBV, HCV and HIV infection.
A prevention plan is mandatory to minimize the risk of accidents; for this purpose every laboratory should perform a risk assessment in order to evaluate the risks that could be encountered inside the laboratory. Risk assessment must take in account every material, every procedure, workers, environment and the different protective equipment. In the context of laboratory accidents, personnel training plays a key role: the workers are directly exposed to risk in case of emergency and they are the first operators involved in emergency maneuvers.

Biohazardous Waste Management
Martien Broekhuijsen

Biohazardous waste (biowaste) is any waste (solid, liquid, airborn) that contains (potentially) biohazardous materials. Biowaste is unavoidably produced in laboratories where work with biohazardous materials is conducted. International and local legislation contain specific guidelines for dealing with biowaste. Several manufacturers produce equipment for destruction, containers for collection, or chemicals for inactivation of biowaste.
Several terms are relevant in this area: Destruction, inactivation, disinfection, decontamination, etc. All methods or procedures are aimed at rendering the material harmless for humans and the environment, or at least to significantly reduce the risk. Methods include incineration, chemical decontamination, autoclaving, irradiation, and other methods.
Validation or verification of any method is important in controlling the biohazard. Some biological materials are harder to inactivate than others. For example, spores of Bacillus anthracis (causative agent of Anthrax) are not affected by some methods that completely inactivate bacterial cells. To demonstrate effectiveness, bio-indicators (e.g. containing spores of Geobacillus stearothermophilus) can be used.
The choice of inactivation method or the way in which biowaste is handled greatly depends on the volume of the waste, the biohazard content (risk level), and practical considerations including economic factors. In the end, any method must have been validated.
If biowaste is transported to another facility for final disposal, then the transport itself poses a risk factor which must be addressed. In such cases, verification and chain of custody are necessary.

Legislation and Guidelines
Martien Broekhuijsen

Legislation for work with biohazardous materials is in existence for many years. Most developed countries have a framework of legislation for such work at a national level, including specific authorities and inspection bodies. Within the EU, all countries also fall under the European biosafety legislation. The biosafety rules that apply in the US and Canada are often considered in the EU as valuable resources for comparison, even up to local decisions to fully implement those rules. A laboratory or research facility that operates at an international level should indeed do well to follow the North-American guidelines as well as the required European rules. For a large part there is extensive overlap between the various guidelines.
A basic distinction can be made between legislation for work with naturally occurring hazardous biological agents (BA) and genetically modified organisms (GMO). Although risk assessment, risk levels, and safety measures are often comparable between work with either BA or GMO, there can be very relevant distinctions. In the EU for example, introduction of GMO in the environment is in principle not permitted, whereas in the US this less difficult to accomplish.
As an example of different national legislation for BA and GMO, the Dutch rules for work with BA or GMO fall under the authority of two different ministries, as BA are predominantly seen as a hazard to workers (focusing on worker protection) and GMO predominantly as a hazard to the environment (focusing on containment). At the EU level, there are specific guidelines for BA[1] and for GMO[2].
Specific legislation exists for transport of hazardous biological materials, e.g. the Dangerous Goods Regulations of IATA[3]. Additional legislation might exist for publication of knowledge, dual use, import and export, use in environment (especially for GMO), and use in agriculture or food. Any organisation working with hazardous biological materials should ensure that all applicable legislation is known and implemented.
It is important to appreciate that legislation and guidelines can (and most likely will) change over time. This can be caused by new knowledge of biohazard, evolving insight, or newly discovered pathogens. Another example is the consequence of the planned eradication of poliovirus, whereby the WHO has already issued plans[4] for stricter safety rules for work with poliovirus.

[1] EU directive 2000/54/EC on protection of workers from risks related to exposure to biological agents
[2] EU directive 2009/41/EC on the contained use of genetically modified micro-organisms
[3] http://www.iata.org/publications/dgr/Pages/index.aspx
[4] http://www.polioeradication.org/Portals/0/Document/Resources/PostEradication/GAPIII_2014.pdf

Accreditation and Certification
Martien Broekhuijsen

Accreditation is the process in which certification of competency, authority, or credibility is presented[1]. Usually, a third (fully independent) party evaluates a product, process, or organisation against specific requirements. If compliancy is concluded, the third party issues an official written statement (certificate) to confirm this.
Certification is a more loose term that refers to the confirmation of certain characteristics of an object, person, or organisation[2]. Usually this is accomplished by some form of external review or audit. Any person or organisation can certify a product, process, or other organisation, but the value of the certificate issued is greatly depending on the status of the certifying body.
Organisations that issue certificates are often in turn accredited by official accreditation bodies. Accreditation bodies are established in many countries with the primary purpose of ensuring that conformity assessment bodies are subject to oversight by an authoritative body[3].
Any certification is dependent on the stated requirements. It does not state an absolute level of competency, but rather establishes compliancy of the product, process, or organisation with a written set of requirements. Compliancy is not meaningful if the requirements are not clearly described. Certification must clearly mention which requirements were evaluated in order to confirm compliancy. In many cases certification is based on internationally established standards such as ISO.

[1] https://en.wikipedia.org/wiki/Accreditation
[2] https://en.wikipedia.org/wiki/Certification
[3] International Accreditation Forum (IAF), http://www.iaf.nu

Biosafety Level 2
Cristina Pagani

Biosafety Level 2 facility is applicable to clinical, diagnostic and teaching laboratory and is suitable for work involving agents of moderate individual risk and low community risk (eg. Salmonella sp. Hepatitis B virus, HIV, Toxoplasma spp.).
Secondary barriers to reduce potential environmental contamination are required, such as sinks for washing hands and waste decontamination facility.
It differs from BSL-1 because the laboratory personnel has specific training in handling pathogenic agents receiving annual update or additional training as necessary for procedural or policy changes. Moreover persons who are at increased risk of acquiring infection, or for whom infection may have serious consequences are not allowed in the laboratory.
The access to the laboratory is limited when work is being conducted, precautions are taken with contaminated sharp items, and procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets. Personal protective equipment as gloves, laboratory coats, goggles, mask and face shield must always be worn while in the laboratory. This protective clothing must be removed and left in the laboratory before leaving for non-laboratory areas.

BSL3 - Agents
Sara Rimoldi

Microorganisms are internationally classified by a risk group (1,2,3,4).
Different microorganisms (viruses and bacteria) are reported as high priority agents that pose a threat to national security. They can be easily disseminated or transmitted person-to-person, cause high mortality, with potential for major public health impact, might cause panic and social disruption and require special public health preparedness.
For their diagnosis different methods are used: microscopy, molecular tests, antigens and antibodies. Cell culture test is used only for viruses such as culture is involved in the bacteria diagnosis.

BSL3 – Specificities
Alessandro Mancon

BSL 3 agents pose a risk for operators and communities. For this reason, their manipulation requires dedicated measures, in order to minimize the occurrence of laboratory acquired infections and the spread in the population. Different national and international institutions (i.e.: WHO, CDC) provide indications to properly work with these agents.
BSL 3 facilities can serve both research and diagnostic purposes; obviously, differences related to these aims exist, mainly concerning the use of animal models.
BSL 3 laboratories are enclosed environments: air and fluids systems are dedicated and not shared with other areas; moreover, only authorized personnel can enter. Such a condition can be obtained with an engineering planning before the construction building phase. Regarding the work in the laboratory, specific rules indicate which standards that must be followed as regards: Personal Protective Equipment, safety cabinets, decontamination agents, work procedures. Also emergency situations must be considered: they need particular measures, not adopted in daily routine activity.
In the Laboratory of Clinical Microbiology, Virology and Bioemergencies (CLIMVIB) at Luigi Sacco Hospital, a BSL 3 facility is present: its use is strictly related to diagnostic purpose, without any animal specificity. The examples presented refer to clinical experience and depict a real-life situation: Biosafety rules and indications are adapted to CLIMVIB needs and they could differ from other institutions. However, all institutions must meet Biosafety principles, in order to prevent personnel infections, epidemic and/or pandemic and related panic in the communities.

BSL 4 - Agents
Romualdo Grande

BSL4 agents are infectious microorganisms transmitted human by human by several routes from direct contact to breathing an aerosol suspension. This infectious microorganisms are very harmful and can cause dangerous diseases for humans due to their aggressiveness and high pathogenicity. Moreover, up to these days, there are no drugs, neither preventive profilaxis able to contrast the undesired effects of these agents. Taking into account the above mentioned characteristics of these agents, we need to answer two important questions: how to protect the workers that and for the human society.
In Italy a list of Level 4 bioagents was licensed and defined in D.L. 81/2008, the national law ruling prevention and safety on work. American CDC listed these microorganisms in his Biosafety in Microbiological and Biomedical Laboratories manual and many others International Boards did the same.
All these microorganisms are virus and all but one are etiological agents of Zoonoses. Variola virus only is specific for man and get no animal reservoir. It was declared extinguished in the late 70’ of the last century but now we have strains stored in U.S.A. facilities and in other facilities of former Soviet Union and the lasts are source of concern for a criminal misuse.
Ebolavirus is the most famous Level 4 agent at this time. It’s a zoonotical agent but his animal reservoir is multiple, involving different animal species and not all are well identified.
It’s the etiological agent of the recent outbreak in West Africa with many thousands of casualties. In spite of his zoonotic origin (in all Ebolavirus outbreak the index case was a zoonosis) can easily infect man to man by direct contact.
The latest outbreak of Ebola virus disease in the Guinea Gulf countries, represents the biggest outbreak by a BSL4 agent, if we don’t consider the Variola plagues of the past centuries.
The other Level 4 virus, by contrast, as Lassavirus, Nipahvirus, CrimeaCongovirus have caused, until now, spotted outbreaks.
In this lesson we’ll examine the different characteristics among the virus belonging to this pathogen class.

BSL4 - Specificities
Davide Mileto

The facilities of biosafety level 4 (BSL4) are designed in order to manage biological agents belonging to risk group 4 and patients infected by such kind of agents.
The main feature of this facility is related with the need to avoid the infection of workers but also with the requirement to prevent the spread of the level 4 biological agents into the environment.
Different international guidelines (WHO or CDC guidelines) describe all the specificities and the correct standards of behavior to be adopted for the BSL4 facilities.
Among them surely the need to have always almost two workers inside of the laboratory and an external support team outside of the laboratory. Communication between the two different teams, internal and external of the laboratory, is mandatory in order to monitor all the different steps and procedures and to act in case of emergency. Laboratory protective clothing must be of the type with solid-front or wrap-around gowns, scrub suits, coveralls, head covering and, where appropriate, shoe covers or dedicated shoes. All the personal protective equipment must be decontaminated before being disposed of. The complexity of the structure makes it mandatory to organize periodic training also to cope with accidents or illnesses.
Different technical features are need for the BSL4 laboratory.
A negative pressure system inside the laboratory guarantees the primary containment. This condition is also provided thanks to the system of interlocked door necessary to enter and exit from the laboratory. The biosafety cabinet of level III are used inside of the laboratory and supplies and materials are introduced through a double-door autoclave or fumigation chamber. A HEPA filter system cleans incoming and outgoing air flows including the air flow need to work with the positive pressure suit. All effluents from the suit area, decontamination chamber, decontamination shower, or class III biological safety cabinet must be decontaminated before final discharge. Emergency power and dedicated power supply lines must be provided.