LECTURE TIMETABLE
TRAINING COURSE BROCHURE

Lecturers

Martien Broekhuijsen

Romualdo Grande

Alessandro Mancon

Davide Mileto

Angelo Moretto

Gijsbert van Willigen

Tutors

Giovanna Fotia

Mirko Palestrino

Please download the registration form here.

Laboratory Biosafety
Martien Broekhuijsen

Laboratory Biosecurity, Dual Use of Biological Materials and Ethical Issues
Gijsbert Van Willigen

Laboratory biosafety describes containment principles, technologies, and practices to protect people from biological agents, and prevent accidental release of biological agents. In addition to biosafety, laboratory biosecurity measures aim to prevent theft and intentional or malicious use of biological agents. Thus, both biosafety and biosecurity should be an integral part of program management of organizations handling dangerous pathogens, in order to prevent potential dual-use research, undesired spread, theft, malicious use, and bioterrorism.
A well implemented laboratory biosecurity program should be implemented. Dual-use biological agents can be used in bioterrorism. Dual-use good are materials that have a civilian and a military application. For these dual-use organisms a stringent biosecurity regime should be in place. Besides the wild type agent themselves, biological agents can also be modified in the lab to become more dangerous than the wild-type organism itself. This is called dual-use research of concern (DURC). One type of DURC research id gain-of-function research of GOF.
Besides the risks of dual-use agents and DURC/GOF also ethical questions can arise from the application of GOF.
The lecture will focus on:
- what should be part of a proper laboratory biosecurity program and who are stakeholders in the program;
- what are dual-use materials;
- what are dual-use biological agents are and the basis why agents are classified as dual-use. The criteria include besides the pathogenicity also other factor such a how can they be cultured and dispersed;
- types of research will be presented as examples of DURC/GOF and published examples will be discussed;
- ethical questions.

Key Reference Reading:
Simon Whitby et al. Preventing Biological Threats: What You Can Do, (Bradford: University of Bradford, 2016).
Tatyana Novossiolova, Biological Security Education Handbook: The Power of Team-Based Learning, (Bradford: University of Bradford, 2016).[1]

[1] Both books are freely available at: http://www.bradford.ac.uk/social-sciences/peacestudies/research/publications-and-projects/guide-to-biological-security-issues/

Regulatory Aspects
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/

Disinfection, Sterilization and Decontamination
Gijsbert van Willigen

For inactivation of biohazardous material, including biohazardous waste, a large number of methods are available ranging from chemical inactivation using chemical disinfectants to various types of a physical process, such as heat for inactivation. All methods aim at rendering the biohazardous material harmless for humans, animals and the environment. In the presentation a large numbers of methods for disinfection, sterilization and decontamination will be discussed that can be used in a laboratory setting together with their advantages and disadvantages for use.
The method of choice for inactivation of biohazardous materials greatly depends on the composition, volume and risk level of the biological material, but also if the material that is being decontaminated is reusable or disposable. Also economic factors can be considered when choosing an inactivation method. After a method is chosen the inactivation process must be validated.
For a proper disinfection, sterilization and decontamination, validation and verification of the processes should be in place. For this several methods can be used to demonstrate the effectiveness of the process depending on the method used. This ranges from biological tests to the use of biological indicators when physical processes are used for inactivation and measuring physical parameters of the process. In the presentation pros and cons of these methods will be discussed.

Biohazardous Waste Management
Gijsbert Van Willigen

Solid (including sharps) or liquid biohazardous waste is unavoidably produced in facilities where laboratory experiments, experiments with animals are performed during the treatment of patients. For save handling of this waste it should be collected in such a way that it does not pose any risk for the people who handle or transport this waste inside or outside of the institute. Special bags, over packs and containers should be used for safe collection of biohazardous waste. They should be leak and puncture proof and can be used for various types of decontamination. Preferably they cannot be reopened. For biohazardous waste national and international legislation is in place for dealing with biohazardous waste.
The presentation will focus on the collection, storage and transport of biohazardous waste. Also some of the commonly made mistakes will be shown. Waste treatment i.e. inactivation will be the topic of the lecture on disinfection, sterilization and decontamination and will only be briefly touched in this lecture.

Practical exercises with fluorescent markers and UV lights
Gijsbert Van Willigen, Martien Broekhuijsen

Purposes of the exercises:
1. To see if hand washing was done thoroughly.
2. To demonstrate how easy a contamination occurs.
3. To see if surface cleaning was done thoroughly, or see effect of different cleaning methods.
Further details were provided during the exercises.

Laboratory Accidents
Davide Mileto

The security of work place is one of the most important aspects of the working activity. The risk assessment for the scientific laboratory, chemical and biological, is linked with the presence of hazardous factors present in the daily lab activities. Among the different elements to be considered in a risk assessment, there are the agents, the equipment, the problems linked with the space limitations, organizational/management aspects and sometimes the lack of information, education and training of the staff. The presence of these factors in a laboratory setting could cause laboratory accidents, such as break of tubes in a centrifuge, projection of liquid in eyes, spills, accidental injection of a contaminated solution, aerosol of liquid solution that could generate the spread of agents into the laboratory environment thereby causing the infection of workers or the contamination of equipment and materials, etc. Accordingly, a relevant problem in the context of the laboratory accidents is all infection 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, the bloodborne Pathogens are important especially for HBV, HCV and HIV infections.
A prevention plan is mandatory to minimize the risk of accidents. Every laboratory should perform a risk assessment in order to evaluate which are the risks that could be encountered inside the laboratory. Risk assessment must take in account every material, every procedures, 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.

Laboratory Acquired Infections
Romualdo Grande

Laboratory-acquired infections due to a wide variety of bacteria, viruses, fungi, and parasites have been described. Although the precise risk of infection after an exposure remains poorly defined, surveys of laboratory-acquired infections suggest that Brucella species, Shigella species, Salmonella species, Mycobacterium tuberculosis, and Neisseria meningitidis are the most common causes. Infections due to the bloodborne pathogens (hepatitis B virus, hepatitis C virus, and human immunodeficiency virus) remain the most common reported viral infections, whereas the dimorphic fungi are responsible for the greatest number of fungal infections. Because of the increasing attention on the role of the laboratory in bioterrorism preparation, I discuss the risk of laboratory-acquired infection with uncommon agents, such as Francisella tularensis and Bacillus anthracis. Physicians who care for a sick laboratory worker need to consider the likelihood of an occupationally acquired infection while advising exposed laboratory workers about postexposure prophylaxis. In addition, physicians should be aware of the importance of alerting the laboratory if infection with a high-risk agent is suspected.

Introduction to Occupational Medicine
Angelo Moretto

The aims of occupational medicine are the identification of health risks in the workplace, health surveillance of workers, diagnosis and treatment of occupational diseases, and in collaboration with professionals with other expertise (e.g. engineers, chemists, industrial hygienists, psychologists) participation to training programs and activities of risk management measures. Over the year, a shift has been observed in the attitude towards health and safety in the workplace. From the XVIII century Bernardino Ramazzini’s first textbook on occupational diseases to the modern legislation, the active involvement of the workers has been increased. In fact, in the XXI century workers are required to actively participate in the management of health and safety in the workplace. This has two consequences: on one hand workers are entitled to provide suggestions and participate in the planning of the activities related to their health and safety through their representatives; on the other hand workers also became responsible and liable of their behaviour with respect to their and their co-workers’ health and safety.
Accidents (injuries) in the workplace, and occupational or work-related diseases are taken care of by specific insurance schemes, that may differ between countries, even within the European Union. According to the Organisation of Economic Co-operation and Development (OECD) “An occupational injury is any personal injury, disease or death resulting from an occupational accident; an occupational injury is therefore distinct from an occupational disease, which is a disease contracted as a result of an exposure over a period of time to risk factors arising from work activity”. Instead, the term occupational disease “is linked to the identification of a specific cause-effect relationship between a harmful agent and the affected human organism. However, it is not easy – and considerably more difficult than in the case of accidents – to prove that a disease is occupationally conditioned, i.e. caused by conditions at, not outside work”. Because of the difficulty in proving a disease to be occupational in origin, most countries have produced lists of prescribed occupational diseases. These are generally limited to those diseases where a strong cause-effect relationship has been proven. However, with the number of categories ranging from 50 to 90, national lists vary in terms of those diseases recognised as occupational. Recommended lists developed by the International Labour Organisation and the European Communities seem to have led only to limited degree of harmonisation. In addition, in certain countries, including Italy, the list is not prescriptive, and any worker can claim a disease as occupational or work-related provided that she/he is able to prove the causal relationship. Each country has different legislative approaches related to registration, notification, and compensation for occupational injuries or diseases and their health consequences.
The risks related to exposure to biological agents at work are subjects to an EU directive (2000/54/EC of 18 September 2025). This Directive has general provisions that have been implemented by local legislation in EU member Countries. General provisions of the directive include the definitions and the assessment of risks. Employers’ obligations including replacement, reduction of risks, information to authority, provision of adequate hygiene and individual protection devices, information and training of workers, compilation of a list exposed workers, active participation of workers, and notification to the competent authority. In addition, health surveillance, measures for the workplace, classification of biological agents based on their characteristics. The class of the biological agent will determine the different provisions that should be taken to protect the health and safety of the workers, and the consequent administrative, technical, preventive and sanitary measures.

BSL3: Agents & Specificities
Alessandro Mancon

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.
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 among 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 ASST Fatebenefratelli Sacco, 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. Naturally, each institution must meet Biosafety principles, in order to prevent personnel infections, epidemic and/or pandemic and related panic in the communities.

BSL4: Agents & Specificities
Romualdo Grande, 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 needed 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 the laboratory whereas 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.

HOW TO REACH L. SACCO UNIVERSITY HOSPITAL

Via G.B. Grassi 74
20157 Milano

Railroad link
Stop Milano Certosa (2 km distant to L. Sacco University Hospital)
-Suburban line S5 (Varese – Treviglio )
-Suburban line S6 (Novara – Treviglio)

Trams
-Line 12, 19 (end of line in front of L.Sacco University Hospital)

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