BIOSAFETY MANUAL First Edition 2016

Transcription

1 BIOSAFETY MANUAL First Edition 2016

2 CONTENTS PAGES I. FOREWORD 3 II. PART I: GENERAL GUIDELINES 4 A. DEFINITIONS 4 B. PRINCIPLES OF BIOSAFETY 4 1. LABORATORY PRACTICES AND TECHNIQUES 4 2. TRAINING 5 3. BASIC REQUIREMENTS FOR WASTE MANAGEMENT 5 4. FOUR BIOLOGICAL SAFETY LEVELS 7 i. BIOLOGICAL SAFETY LEVEL 1 (BSL1) 7 ii. BIOLOGICAL SAFETY LEVEL 2 (BSL2) 7 iii. BIOLOGICAL SAFETY LEVEL 3 (BSL3) 8 iv. BIOLOGICAL SAFETY LEVEL 4 (BSL4) 8 5. BIOLOGICAL RISK ASSESSMENT 9 III. PART II: LABORATORY EQUIPEMENT 11 A. BIOLOGICAL SAFETY CABINETS CLASS I BIOLOGICAL SAFETY CABINET CLASS II BIOLOGICAL SAFETY CABINET CLASS III BIOLOGICAL SAFETY CABINET 14 B. OTHER SAFETY EQUIPEMENT PERSONAL PROTECTIVE EQUIPMENT AND CLOTHING PIPETTING UTILITIES HOMOGENIZERS, SHAKERS, BLENDERS, SONICATORS DISPOSABLE/ ONE USE LOOPS LEAK PROOF CONTAINERS FOR COLLECTION AND TRANSPORT SHARP DISPOSAL CONTAINERS AUTOCLAVE SCREW CAPPED TUBES AND BOTTLES NEGATIVE PRESSURE FLEXIBLE FILM ISOLATORS 17 IV. PART III: STANDARD LABORATORY TECHNIQUES 18 A. HANDLING OF SPECIMENS 18 B. SAFE USE OF PIPETTES AND PIPETTING AIDS 18 C. HANDLING SHARP MATERIALS AND NEEDLES 19 D. PROPER USE OF BIOLOGICAL SAFETY CABINETS 19 E. STANDARD MICROBIOLOGICAL PRACTICES 19 F. PREVENTING THE SPREAD OF BLOODBORNE PATHOGENS 20 V. PART IV: SURVEILLANCE AND OCCUPATIONAL HEALTH 21 VI. PART V: INTRODUCTION TO BIOSECURITY 21 A. GENERAL BIOSECURITY GUIDELINES VALUABLE BIOLOGICAL MATERIALS (VBM) DUAL USE PURPOSES LABORATORY BIOSECURITY MEASURES 23 VII. PART VI: INTRODUCTION TO BIOETHICS 24 VIII. REFERENCES

3 I. FOREWORD Information concerning the biosafety procedures utilized by laboratories in Lebanon and included in the manual came in part from individuals who participated in the conferences sponsored by the Lebanese Association for Biosafety, Biosecurity and Bioethics (LABBB). They included Drs. Bassem Shabb, Jacques Mokhbat, Fayez Abillamah, Abdelrahman Bizri, Abdo Jurjus, Pierre Abi-Hanna, Adel Mastori and Mr. Talal Abou Mjahed. Other ideas came from already established manuals that can be found on the WEB including National Institute of Health (NIH), Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO). An Arabic translation of the manual was also prepared. The preparation of this manual was fully supported by CRDF Global and Biosecurity Engagement Program (BEP) for which we are grateful. Alexander M. Abdelnoor, BS Pharm., MS, Ph.D. President of LABBB Professor and Chairperson Department of Experimental Pathology, Immunology & Microbiology Faculty of Medicine American University of Beirut Beirut, Lebanon Nayla S. Al Akl, MS Secretary of LABBB Senior Technician Department of Experimental Pathology, Immunology & Microbiology Faculty of Medicine American University of Beirut Beirut, Lebanon LABBB All rights reserved. Requests for permission to reproduce or translate LABBB publications whether for sale or for noncommercial distribution should be addressed to Dr. Alexander Abdelnoor Abdelnoor. LABBB does not warrant that the information contained in this publication is complete and correct and shall not be liable for any damages incurred as a result of its use. Printed, Beirut, Lebanon - 3 -

4 II. PART I: GENERAL GUIDELINES A. DEFINITIONS Biosafety: is the prevention of accidental release of hazardous infectious and/or toxic agents into the laboratory setting and environment. It is maintained by regular review and application of biosafety guidelines and containment principles. Biosecurity: is the set of assessment procedures in addition to the measures adopted to study the probability of intentional misuse of hazardous biological material that could harm the environment and society. It protects against the misuse of science in today s world. Bioethics: is the study of ethical conduct, recording and reporting results in biological research and medicine in general. B. PRINCIPLES OF BIOSAFETY Biosafety is mandatory in the containment of possibly hazardous biological agents. It is achieved by implementing safe procedures for handling infectious material, defining facility design and equipment needed for safe management of these agents in any laboratory environment. The purpose of biosafety guidelines is to contain infectious agent spread, such as accidental exposure or accidental release, by decreasing or eliminating exposure of laboratory workers, other individuals as well as the external environment. The level of biosafety required for each infectious agent is defined after thorough risk assessment of the work to be implemented with the particular agent. 1. Laboratory practices and techniques Scientists are accountable for the conduct of their work when handling any infectious agent or product and for applying appropriate laboratory and safety procedures. Proper biosafety practices include: - Adherence to standard microbiological practices and good microbiological techniques (GMT) should be applied to ensure efficient containment. (GMT adopted from WHO and explained in sections below) - Training of laboratory personnel should be arranged and provided by the laboratory director or supervisor. Training could be assisted by the biosafety professionals or officers. - Laboratory personnel or anyone handling the infectious agent of concern should be well aware of any potential hazards imposed by the agent at hand in addition to being capable of conducting the required practices and techniques safely and proficiently. - Risk assessment should be conducted to identify the hazardous features of the infectious agent manipulated. - Personnel should consult with biosafety professionals with regard to risk assessment in planning their work. - A guiding biosafety manual should be available to help detect the potential hazards that could be encountered and identify the processes to be employed to diminish or exclude exposures to these hazards

5 - Meeting the requirement for safe laboratory practices and procedures should be accompanied by proper facility design, safety equipment, and competent management practices. 2. Training A key element in the prevention of accidental spread of infectious or hazardous agents is personnel training. A staff that is well aware of the appropriate recognition and handling of hazardous material is a vital milestone in safeguarding biosafety and preventing incidents or accidents. Training is carried out by professional personnel such as the biosafety officers or by the laboratory director or both. It should cover: - Information on the correct use of equipment i.e. standard operating procedures (SOP) of machines, signing of user log sheets, routine inspections, potential errors, and regular recertification of equipment. - Proper techniques and practices on safe ways for conducting high-risk hazardous procedures that are faced by laboratory members. - The mandatory use of Personal Protective equipment (PPE) whenever in the laboratory. Laboratory members should also be trained on the different types of PPE and their corresponding use. - Prevention or protection from production of aerosols that can pose a serious inhalation risk. Practices that pose aerosol production risk include pipetting (especially mouth pipetting), preparation of smears, centrifugation, streaking and handling agar plates, use of loops and others without proper ventilation and necessary masks/respirators and/or safety equipment. - Prevention or protection from ingestion risk, such as swallowing or mouth contact when handling specimens, smears or cultures. Mouth pipetting should be avoided. The urgency to change and properly dispose of contaminated gloves as required should be explained to laboratory members in addition to the necessity to avoid contaminating surfaces that could pose risk for other staff members. - Proper use and disposal of syringes and needles to prevent percutaneous exposure. - Correct handling and transport of microbiological or pathological specimens and blood. - Decontamination and proper discarding of hazardous and infectious material. Training sessions are a continuous process, whenever new members join the lab, or new safety measures are installed. Sessions should be administered regularly to insure awareness and adherence. 3. Basic requirements for waste management The principle of waste management is to secure that all items being handled in the laboratory are clean and do not pose a risk of infectious agent exposure to the laboratory workers and outside environment. Waste products are defined as anything to be discarded; however, in daily laboratory work not all items can be discarded as some of them are reused or recycled after appropriate treatment. For the sake of this section, the term waste will include both types, waste for discard or reuse

6 In order to differentiate between the types of waste and their appropriate handling methods, waste should be segregated into categories. These include: - Non-infectious / non-contaminated products that are disposed as general household waste such as cardboard packaging of pipettes, paper, wrapping paper, etc. Laboratory clothing should be washed and cleaned regularly. - Contaminated material that can be reused or recycled. These items include laboratory instruments and glassware that should be decontaminated or sterilized and thereafter washed for reuse. Pre-cleaning should not be carried out for any contaminated material to be autoclaved and reused. If required, cleaning should take place only after autoclaving and disinfection. - Infectious/contaminated sharps that pose a risk of percutaneous puncture. These items should be collected in appropriate puncture-proof containers. Depending on the waste carried by the sharps, there are two types of sharps containers. Sharps that are contaminated with infectious material are placed in a container that will then be decontaminated and disposed. Sharps that are contaminated with cytotoxic drugs are disposed in containers that are later incinerated. The universal precautions for safe use and disposal of needles and sharps stresses that needles and syringes are used only once. When used, it is important to never recap or bend needles or remove them from disposable syringes, instead the whole apparatus should be disposed in a puncture-proof container clearly labeled Sharps. The disposal container should never be overfilled or reused, they should be incinerated. Syringes could be autoclaved prior to disposal if found necessary. Sharp containers should not be disposed of in landfills. - Contaminated material for disposal are items that should be autoclaved first and then sent for disposal. Autoclaving is done to ensure that infectious materials are killed before sending waste product for disposal thus securing the safety of personnel handling the waste thereafter and the environment. Examples include gloves, agar plates, culture plates, contaminated pipettes, etc. - Contaminated material for direct incineration are products that are not reused and should be incinerated. These include tissues, organs and other biological specimens. THE DIFFERENCE BETWEEN STERILIZATION, DECONTAMINATION AND DISINFECTION Sterilization: The purpose of sterilization is to render any item, device or solution germ free, thus making it sterile. It is defined as completely removing or killing all living microorganisms, including spore-forming bacteria. Disinfection: It is a less lethal procedure than sterilization. It allows the elimination of most, but not all pathogenic microorganisms. Disinfection makes use of physical or chemical means for killing microorganisms but it does not warrant an overkill and therefore it doesn't provide the same level of safety accomplished by sterilization. Decontamination: The purpose of decontaminating laboratory instruments and products is to provide protection to all laboratory members, the general environment and all individuals that are exposed to laboratory products inside or outside the laboratory. It entails use of multiple processes (removing or killing microorganisms) to disinfect work surfaces and equipment

7 4. Four biological safety levels Four different levels of biological safety laboratories (BSL) are identified according to CDC and WHO categorization. Each level is associated with a different risk group of infectious agents. Risk group 1 being the least infectious and group 4 being the most infectious (associated accordingly with BSL 1, 2, 3 and 4). The difference between these laboratories is based on their diverse requirements in practices and techniques, the different safety equipment needed (Primary Barriers) and the facility design (Secondary Barriers). In Lebanon so far, the highest level of biosafety laboratory available is BSL3* present in only one research institution. The requirements for each laboratory associated with its risk group is based on the types of experiments or procedures performed and the nature of the agents manipulated; safety level classification of these agents depends on their route of transmission, potential pathogenicity, stability, concentration and availability of treatment or prophylactic measures. Primary Barriers include both safety equipment and personal protective equipment (PPE); safety equipment such as Biological Safety Cabinets (BSC), enclosed containers, safety centrifuge cups and PPE such as lab coats, gloves, shoe covers, safety glasses, face shields and respirators. Secondary Barriers include the facility construction and design. The focus in the design is to protect the workers and prevent exposure to the outside environment. The choice of design is based on the biosafety level required for the organism being used, the risk of its transmission and accidental release. The barrier design could range from simple separation of laboratory workstation from public access to specialized ventilation systems if the infectious agent can produce aerosols. Each institution, whether medical or research laboratory, should perform risk assessment for the agents expected to be manipulated, the types of procedures/experiments to be performed using the best information available and set up their laboratory accordingly. Laboratory directors are responsible of conducting the risk assessment process, they should provide recommendations in the subsequent training for the rest of the laboratory personnel expected to come in contact or work with these agents. It is advised that institutions recruit a Biosafety Officer that is assigned to overlook the procedures performed and assist the laboratory director in performing the risk assessment and delivering recommendations. The essential requirements for the four BSL laboratories are summarized below. BSL 1 requiring the least safety measure to BSL 4 that builds on all previous levels and more. i. Biological Safety Level 1 (BSL1) BSL1 laboratories include basic teaching and research training laboratories as well as other laboratories that handle microorganisms that are characterized and not known to cause disease in healthy adults. It requires only basic biosafety practices; work is done on an open bench using Good Microbiological methods and PPEs such as coats and gowns to prevent contamination of personal clothing. BSL1 laboratories do not require separation from the general traffic of the building. There are no special secondary barrier requirements other than a sink for hand and eye washing, a door to maintain a controlled access as well as the presence of a nearby autoclave. ii. Biological Safety Level 2 (BSL2) BSL2 laboratories include diagnostic, health care and research laboratories. All safety requirements for BSL1 are used in addition to other protective measures. These types of laboratories have no complete control of the specimens they receive, hence specimens should be handled in a higher safety level and it requires the use of a BSC. Agents manipulated in this laboratory are categorized as moderate hazards that are of no high risk to staff members or the *BSL3 is available at the American University of Beirut - 7 -

8 environment. Awareness should cover mucous membrane accidental exposures as well as percutaneous ones in addition to agent ingestion. If risk of aerosol release is suspected work should always be done within the biological safety cabinet. PPEs should be used as suitable, in addition to gloves, gowns and lab coats, splash shields and face protection. Standard operating procedures should be well defined and recognized by all laboratory personnel. Specific training should be given to all personnel for handling disease-causing agents, and work should be supervised by knowledgeable scientists. The primary barriers needed other than the Biological Safety Cabinet are the availability of an autoclave; it is preferable to have an autoclave on site, otherwise, an autoclave should be available nearby in the facility. Concerning secondary barriers, it is preferable to design an inward airflow and controlled ventilation system in the laboratory to prevent contamination of specimens and the release of pathogens to the outside. Restricted laboratory access should be maintained especially when work is being performed. iii. Biological Safety Level 3 (BSL3) BSL3 should be used for research, teaching, diagnostic, clinical and production laboratories that manipulate highly pathogenic organisms known to cause serious infectious diseases if spread through aerosol. Respiratory transmission is the main concern in providing protection with BSL3 laboratories as it may cause grave and potentially deadly infections. All basic requirements for BSL2 are applied in addition to specific guidelines to meet the biological safety level 3. Primary barriers include the use of a biological safety cabinet (optimally class II or III) and other physical containment agents, such as a gas-tight aerosol chamber and centrifuge cups; in addition to personal protective clothing that should never be worn outside the laboratory, it is preferable to use solid front gowns. Clothing must be changed when contaminated. Concerning facility design, these types of laboratories should be isolated from unrestricted traffic areas and should have controlled access. Room must be sealable in order to achieve decontamination and double entry doors must be used. In addition, inward airflow and a controlled ventilation system should be employed to sustain the pressure differential between the laboratory and neighboring spaces and prevent aerosol release to the outside environment. HEPA filtered air exhaust, anteroom and an on-site autoclave must also be available. A hands-free sink, used specifically for handwashing, should be provided near the exit door. iv. Biological Safety Level 4 (BSL4) The information given in this manual on BSL4 is intended as introductory for BSL4 set up. Facilities aiming to develop such a system should contact the WHO Biosafety program for supplementary information. Set up and operation of maximum containment laboratories should be under the control of national or health authorities. The highest level for containment laboratory is BSL4. Infectious agents manipulated in this type of laboratory are known to pose a high risk of fatal disease. Such agents are potentially transmittable via aerosol production and no vaccines or treatment are available. Agents that are not yet identified and have a highly similar or identical antigenicity to BSL4 agents should be maintained and manipulated at this level until enough information has been gathered in order to categorize them in the appropriate safety level laboratory. The primary hazard to laboratory staff is risk of respiratory and mucous membrane exposure. The main focus in a BSL4 is to maintain a strict isolation of aerosolized contagious agents or associated material. This is achieved by using the appropriate primary barriers, such using a Class III BSC for conducting work or alternatively employing a full-body, air-supplied positive-pressure personnel suit. An autoclave must be present in the laboratory and must have double-ended doors

9 Requirements for secondary barriers include placing the BSL 4 laboratory in a distinct building, or totally isolated are, with the appropriate ventilation and waste management requirements that encompass a controlled ventilating system, inward airflow, and HEPA-filtered exhaust. Room must be sealable for effective decontamination when necessary. Entrance is restricted to concerned personnel with double-door entry and airlock with shower. Each BSL4 laboratory must have a designated laboratory director that should be in charge of the safe operation of the laboratory, maintaining and safeguarding the safety of the personnel by applying critical knowledge and judgment in assessing and setting up recommendations for safe management of the facility. He/she should provide adequate training for personnel as well. 5. Biological risk assessment Biosafety relies on risk assessment to ensure safety to all laboratory staff, the general public in addition to the environment. The main purpose of risk assessment is to inform the decision making process of the risk at hand and help plan and set up laboratories and procedures to lessen risk of exposure to infectious agents. According to the definition in the Biosafety in Microbiological and Biomedical Laboratories 5th Edition BMBL manual: Risk assessment is a process used to identify the hazardous characteristics of a known infectious or potentially infectious agent or material, the activities that can result in a person s exposure to an agent, the likelihood that such exposure will cause a laboratory associated infection, and the probable consequences of such an infection (3). Risk assessment should be conducted in a timely manner, reviewed routinely and revised when necessary by the laboratory professionals. Several tools are available for these purposes. These tools are most efficient when used concomitantly with professional judgment. Individuals such as the laboratory director, the institution s safety committee* and the biosafety professionals are in charge of conducting the risk assessment on time. They should be familiar with the organism to be handled and its characteristics, the planned procedure to be employed and the available containment equipment and facilities. The result of the assessment will aid in proper selection of the biosafety level required and needed microbiology practices along with necessary primary and secondary barriers. The WHO devised a listing of the risk groups of microbiological agents. It is a tool that aids in the microbiological risk assessment (Table 1). It is based on three main characteristics: the microorganism s infectious and disease-causing capability in a human or animal host, the gravity of the disease(s) caused by the agent and the availability of prophylactic and/or treatment measures. They are divided into 4 risk groups. This table is considered a building stone for risk assessment but is not sufficient as other factors should be considered when conducting risk assessment, because although the four risk groups described below correlate with the 4 different levels of biological safety, they do not equate. A list of specifications for each precise agent will determine the extent of correlation between biosafety level and the risk group classification. The additional factors include (adapted from the WHO Biosafety Manual 3 rd edition): The infectious dose and agent pathogenicity, the natural route of transmission and potential outcome when exposed, accidental exposure routes (percutaneous, ingestion, airborne), agent resilience to environmental conditions, the intended concentration and volume of the agent, host range, type of work proposed (risk of aerosolization), genetic modifications (use of recombinant organisms) that could expand the host range, change its sensitivity or treatment efficacy and finally availability of effective treatment methods. Based on the list and specifications during the risk assessment, agents will be classified to the corresponding biosafety level and the appropriate PPE and SOP * Each institution should develop their own Biosafety committee in order to overlook and approve the work being done at the facility and provide recommendations and certifications when needed.

10 Table1: Classification of infectious agents by risk group (Extracted from the WHO biosafety manual 3 rd edition) Risk Group1 (no or low individual and community risk) A microorganism that is unlikely to cause human or animal disease. Risk Group 2 (moderate individual risk, low community risk) A pathogen that can cause human or animal disease but is unlikely to be serious hazard to laboratory workers, the community, livestock or the environment. Laboratory exposure may cause serious infection, but effective treatment and preventive measures are available and the risk of spread of infection is limited. Risk Group 3 (high individual risk, low community risk) A pathogen that usually causes serious human or animal disease but does not ordinarily spread from one infected individual to another. Effective treatment and preventive measures are available. Risk group 4 (high individual and community risk) A pathogen that usually causes serious human or animal disease and that can be readily transmitted from one individual to another, directly or indirectly. Effective treatment and preventive measures are not usually available. CASES OF SAMPLES WITH INSUFFICIENT DATA In some situations, where limited information is available on specimens delivered into the laboratory, the risk assessment procedures described above are not sufficient. It is advised to treat the sample with caution. Samples should be transported in appropriate sealed containers and should follow international guidelines for transportation of such specimens. Use of PPE is obligatory (gloves, gowns, and eye protection), in addition, samples should be handled at basic containment laboratory levels (BSL2) with use of basic procedures for manipulation; in aims to prevent aerosol release and percutaneous or ingestion risk. For guidance in handling the specimen, information can be found in the medical history chart of patients and in epidemiological data from the ministry of public health. In addition, information can be extracted depending on the geographic origin of the sample. The WHO keeps an updated record on their website in case of outbreaks of infectious agents of unknown etiology Information on communicable diseases can be found in The Control of Communicable Diseases Manual. It provides information on disease severity, susceptibility and resistance in humans in addition to data on transmission modes. In addition, when in doubt guidance from biological safety professionals or experienced colleagues can be sought and is typically helpful

11 III. PART II: LABORATORY EQUIPEMENT A. BIOLOGICAL SAFETY CABINETS The main constituents of primary safety equipment are Biological Safety Cabinets (BSCs). They are intended for providing safety for the laboratory worker, the environment and the work material. The controlled airflow in the cabinets prevents exposure to infectious splashes and aerosols. Several laboratory manipulations allow the production of splashes and aerosols that are not visible by the naked eye such as shaking, pouring, stirring, agar plate streaking, inoculating cell culture dishes, pipetting, centrifugation, vortexing and homogenizing. Proper use of BSC is needed to reduce sample cross contamination and laboratory acquired infections (LAI). The key feature of BSC is the HEPA filter that traps minute particles effectively and only releases organism-free air from the cabinet as exhaust. Choices of BSC to be used are made after thorough risk assessment of the planned work and type of microorganism to be used. Prior to use, biological safety cabinets should be appropriately validated. Recertification should be regularly performed to ensure the cabinets are effective. There are three types of BSCs: 1. Class I Biological Safety Cabinet (BSC I) (Figure1) The first BSC to be introduced was the Class I BSC. This type of cabinet provides protection to laboratory personnel and the environment. This type of BSC allows the drawing in of directional airflow via the front opening. It passes across the work surface and then goes out through the exhaust duct and passes through the HEPA filter for filtration; the air flow transports any workinduced aerosolized particles away from the operator and into the exhaust duct. Since the air drawn in passes over the work area inside the cabinet, this type of BSC does not allow consistent product protection (Figure 1). A glass window is present at the front opening, that permits the operator to observe the cabinet work surface and insert his/her arms to access it. The glass window can be raised and lowered to permit cleaning among other purposes. The exhausted air after passing through the HEPA filter can be released into the outside immediately or by passing through the building exhaust. The exhaust fan can be either present inside the BSC I or in the building. C B D A Side view Figure 1: Schematic Representation of a Biological Safety Cabinet I (Modified from the The Laboratory Biosafety Guidelines: 3 rd edition 2004, Public Health Agency of Canada) A: Front Opening; B: Sash; C: Exhaust HEPA Filter; D: Exhaust plenum

12 2. Class II Biological Safety Cabinet (BSC II) The Class II BSC were developed to allow protection of the sample from contaminated room air, since Class I BSC provided protection only to personnel and environment while flowing inward air is contaminated and thus risky to flow across the work surface. Only the air filtered through the HEPA filter flows across the materials inside the cabinet. These types of cabinets are designed for procedures involving pathogens in containment laboratory levels 2 and 3 and level 4 only when positive-pressure suits are used. There are four types of Class II BSCs. The differences between the BSCs relies on several aspects: the velocity of the air drawn inside the cabinet, the percentage of recirculated versus exhausted air, the exhaust system that either recirculates air into the room or to the outside, and the pressure arrangements of the cabinet ducts and plenums. Class II, Type A1 Cabinets (Figure 2): Ambient air is drawn into the cabinet by an inner fan through the opening (velocity 0.38m/s) and passes through the front intake grill to flow through the supply HEPA filter for sterilization prior to flowing across the work surface. Then the flowing downward air is split and passes from the surface through the front and rear exhaust grills, allowing aerosolized particles to be drawn through the exhaust grill, thereby providing sample and work surface protection. The air is then cleared via the rear plenum to the area between the exhaust and supply filters. The air can either recirculate via the supply HEPA filter to the work surface (70% or pass through the top exhaust filter (30%) to the laboratory or outside. The filtered cabinet air can either circulate back into the laboratory or is channeled out through a dedicated duct to the thimble connection and then outside of the building. This type of cabinets cannot be used in procedures employing low levels of volatile radionuclides or volatile toxic chemicals. C D B E A Side View F Figure 2: Schematic Representation of a Biological Safety Cabinet II type A1 (Modified from The Laboratory Biosafety Guidelines: 3 rd edition 2004, Public Health Agency of Canada) A: Front Opening; B: Sash; C: Exhaust HEPA Filter; D: Rear plenum; E: Supply HEPA filter, F: Blower Class II, Type A2 Cabinets (Figure 3): The Class II Type A2 cabinets are a variation of the Class II Type A1. It has the same functioning capacity as Type A2 but the different features are in the air drawn inside the cabinet that sustains a minimum average face velocity of 0.5 m/s and the presence of a duct at the upper exit and plenum

13 that functions under negative pressure. This type of cabinets can be used in procedures employing low levels of volatile radionuclides or volatile toxic chemicals. F C E B D A Side View Figure 3: Schematic Representation of a Biological Safety Cabinet II type A2 (Modified from The Laboratory Biosafety Guidelines: 3 rd edition 2004, Public Health Agency of Canada) A: Front Opening; B: Sash; C: Exhaust HEPA Filter; D: Rear plenum; E: Supply HEPA filter, F: Duct Class II, Type B1 Cabinets (Figure 4): The class II, Type B1 cabinet is another variation of Class II BSC. The main feature of this type of cabinet is that it possesses a hard duct that releases air into the atmosphere upon passage via the HEPA Filter. Only 30% of the filtered air goes back into circulation and 70% is exhausted to the outside. Additionally, this type of cabinets can be used in procedures employing low levels of volatile radionuclides or volatile toxic chemicals. C B D A G E Side View F Figure 4: Schematic Representation of a Biological Safety Cabinet II type B1 (Modified from The Laboratory Biosafety Guidelines: 3 rd edition 2004, Public Health Agency of Canada) A: Front Opening; B: Sash; C: exhaust HEPA Filter; D: Supply HEPA Filter; E: negative pressure exhaust plenum; F: Blower; G: HEPA filter for supply air

14 Class II, Type B2 Cabinets (Figure 5): The main feature of the Class II type B2 cabinet is that air does not recirculate within the cabinet after passage via the HEPA filter, 100% is externally exhausted through a dedicated duct exhaust. Its face velocity is typically around 0.5 m/s. This kind of cabinet is safe for work with radionuclides and volatile toxic chemicals. C D B E A Side View Figure 5: Schematic Representation of a Biological Safety Cabinet II type B2 (Modified from The Laboratory Biosafety Guidelines: 3 rd edition 2004, Public Health Agency of Canada) A: Front Opening; B: Sash; C: Exhaust HEPA Filter; D: Supply HEPA filter, E: Negative pressure exhaust plenum 3. Class III Biological Safety Cabinet (BSC III) (Figure 6) D C E B A Side View Front View Figure 6: Schematic Representation of a Biological Safety Cabinet III. (Modified from The Laboratory Biosafety Guidelines: 3rd edition 2004, Public Health Agency of Canada). A: Glove ports for arm length gloves; B: Sash; C: Double exhaust HEPA Filters; D: Supply HEPA filter; E: Double-ended autoclave or pass-through box; Connection of the cabinet exhaust to an independent building exhaust air system is required

15 Class III cabinets are the most protective type of biological cabinets and are utilized against risk group 4 organisms as they provide the maximum level of laboratory workers and product protection. They are fully enclosed and sealed gas-tight, the in-coming air is HEPA-filtered and the exhausted air is passed through 2 HEPA filters. There is no opening; work is performed through thick long rubber sleeved gloves clutched on the ports of the cabinet. The BSC III is maintained under negative pressure (120Pa) through the ongoing airflow supplied by an exterior dedicated exhaust system. It can be adjunct to a double-ended door autoclave needed to sterilize material entering or exiting the cabinet. A pass through box, equipped with exhaust HEPA filter and can be sterilized, should be attached to the cabinet B. OTHER SAFETY EQUIPMENT Safety equipment consist a major part of the primary safety barriers; they are designed to reduce and/or eliminate certain hazards. When used correctly, at the appropriate biosafety level laboratory with the corresponding approved biological safety cabinet, these equipment can then provide efficient safety against hazardous aerosol release. Protection is not ensured if not used correctly; laboratory workers should be trained on the proper handling of these equipment. Additionally, equipment should be maintained regularly and tested for their efficiency and safety. Maintenance can be performed by either the laboratory professionals, the medical engineering team or the biosafety professionals. 1. Personal protective equipment and clothing To prevent contamination of personal clothing and protect laboratory workers, PPE is used in all laboratory settings at all times. Their use minimizes the threat of exposure to hazardous spills, splashes, aerosols and unintentional inoculation. A variety of PPE is available for use depending on the type of work to be completed and level of safety required. PPE should only be used when present and actively working in the laboratory, and should be removed before exiting the premises. Handwashing is mandatory before and after working at the laboratory. - Laboratory coats, gowns, coveralls and aprons are used to prevent personal clothing contamination. They should only be worn inside the laboratory along with closed toe shoes to prevent injuries from impact and splash accidents. - Face shields and goggles should be worn provide protection to the face and eyes. Choice of type of protection depends on the procedure planned. Goggles provide side shield protection while face shields cover the entire face. They should only be worn inside the laboratory. - Respirators are used during procedures that encompass high hazard risk, such as washing up a spill of infectious materials. Several filters are available to fit the respirators; they can provide protection against inhalation of aerosols, vapors, fumes, and microorganisms. Choice of respirator to be used depends on the type of expected hazard and can be done with assistance of an occupational hygienist. They should only be worn inside the laboratory. Surgical masks do not provide respiratory protection to laboratory workers. - Gloves are used to protect and prevent hand contamination during any laboratory procedure. They are available as disposable vinyl, nitrile or latex medical approved gloves. They should be used for all-purpose laboratory work and for manipulating any infectious material or blood and bodily fluids. Recyclable gloves are also acceptable for work but care must be taken for the appropriate washing and disinfection. Gloves should be changed when handling different materials and should be removed after working with infectious products or in a biological safety cabinet. Handwashing is mandatory after gloves removal

16 2. Pipetting utilities Pipetting procedures must always be performed with a pipetting aid; pipetting through mouth poses increased hazardous risk and thus should be strictly prohibited. Most common accidents for hazard exposure are attributed to mouth pipetting such as ingestion of pathogen, aerosol inhalation due to suction on pipette and dripping from pipette. Several designs of pipetting aids are available and they include several features, such as ease of use, option to be sterilized, leakage control and control of contamination as well as protection of user. The inhalation of aerosols may be unavoidable during pipetting operations especially when the last drop is blown out, the risk can be diminished by conducting the work inside a biological safety cabinet. 3. Homogenizers, shakers, blenders and sonicators Homogenizers, shakers, blenders and sonicators to use at laboratory set-ups should be designed to fit that purpose, since regular domestic machines may not be sealed and can cause aerosol release. If homogenizers are used for high hazardous risk microorganisms, they should always be operated inside biological safety cabinets. Sonicators can pose a risk of aerosol release. Work with sonicators should be performed inside the biological safety cabinet, and if a cabinet is not available then sonicators should be shielded. The shields and exterior surfaces of sonicators should be disinfected after use. 4. Disposable/ One-Use loops Disposable/ One-use prepackaged loops do not require sterilization before use and eliminate the need to work close to Bunsen burners. Instead, work with this type of loops can be easily done inside a biological safety cabinet, where airflow is not disturbed. When procedure is completed, loops are considered to be contaminated and should be drenched in bleach and discarded as contaminated waste. 5. Leak-proof containers for collection and transport Leak proof containers are essential laboratory equipment; they are needed for collection and transportation of infectious materials since the locked compartment prevents aerosols release, spillage and leakage. They can be used for autoclaving infectious materials as well. 6. Sharp disposal containers Sharps containers are used for disposal of any contaminated sharp products such as needles, blades or broken glass among others. They are puncture-proof plastic containers that can be autoclaved before disposal. Sharps that are contaminated with infectious material are disposed in containers that will then be decontaminated and disposed. Sharps that are contaminated with cytotoxic drugs are disposed in containers that are later incinerated. Containers should be clearly marked as sharps and should never be overfilled beyond the mark indicating volume capacity. Sharp disposal containers should be located with no obstacles or furniture between the place of use and the container. Their location should be easily visible and within close horizontal reach of the worker. Containers should be fixed to walls or other permanent sites; the opening of the container must be easily viewed and accessible to the user

17 7. Autoclave The autoclave is a device designed to sterilize equipment and or biological waste. It is widely used in medical and laboratory facilities to sterilize both reusable equipment and waste products before disposal. Sterilization is based on use of heat, steam and pressure within a chamber or closed vessel. The standard time of the process is at least of 30 min, calculated after the temperature reaches 121 C at a pressure of 15psi. The wet thermal sterilization method is efficient as it uses temperatures high enough to sterilize but low enough to avoid emitting pollutants into the atmosphere. Depending on the biosafety level of the laboratory, autoclaves can be either present nearby in the same facility or on site in the laboratory and could have a double-ended door. Validation of the autoclave must be done before initial operation and recertification should be done regularly. 8. Screw-capped tubes and bottles Screw capped tubes and bottles are small effective container for storage of biological materials. These prevent aerosol release and spillage. 9. Negative Pressure Flexible Film Isolators Negative Pressure Flexible Film Isolators are primary containment devices that offer protection against hazardous infectious materials. They are efficient and flexible for use since they can be mounted on a mobile stand. They are enclosed transparent work spaces made from polyvinylchloride attached to a standing steel framework. Air exhaust and supply is passed through HEPA filters. A negative pressure is conserved within the isolator to deliver protection. Work products can be removed and inserted to the isolator through sample ports. Isolators can be fitted on other laboratory equipment, such incubators and microscopes among others. When manipulating high risk organisms and there is no availability of a conventional safety cabinet, isolators can be used

18 IV. PART III: STANDARD LABORATORY TECHNIQUES Most laboratory accidents and work-related infections are due to human error and improper handling of safety equipment. Most common problems are avoidable. Below is a list of safe practices in the laboratory setting. A. HANDLING OF SPECIMENS To avoid risk of infection in laboratory employees, samples must be handled safely while collected, transported and during procedures. - It is necessary to use appropriate specimen containers that are leak proof, made of glass or alternatively plastic, robust, and capped or closed correctly. The outside of the container should be kept clean with no material on the outer surface. Any container being used in the laboratory should be properly labeled to ensure correct records and safety when handling. Any data sheet tagging with the specimen should come separately in a waterproof envelope and not attached to the container. - Specimens should be transported within the facility using a primary and a secondary container. The primary container is the one previously described and contains the specimen of interest. The secondary container could be an autoclavable box made of metal or plastic and contains racks so the primary containers can stay upright while being transported. Secondary containers should be decontaminated regularly. - The sample receiving area should be a defined zone in the laboratory fitted for receiving large numbers of specimens. Personnel in charge of receiving samples must be aware of any probable hazards involved and should be well skilled to apply standard safety procedures when handling unknown samples or dealing with leaking or broken containers. Primary containers can only be manipulated in a biological safety cabinet. B. SAFE USE OF PIPETTES AND PIPETTING AIDS - Mouth pipetting is strictly prohibited. Pipetting aids must always be used. - Pipettes with cotton plugs are safer to use as they reduce risk of contaminating pipetting devices. Suction of liquid into pipettes should not exceed limit and risk removal of cotton plug and contamination of pipetting aid. When handling infectious material, no force should be applied when blowing out contents as it increases the risk of blowing air and release of infectious aerosols. Avoid mixing infectious material by repeated suction and ejection movements through the pipette. - When work with pipette is finished, contaminated pipettes should be disposed in a appropriate unbreakable antiseptic jar. They should be kept in the disinfectant for the period of time indicated on the label of the corresponding disinfectant that is being used, to insure decontamination before disposal. - It is preferable to cover the work surface with absorbent paper that can be late discarded as infectious waste when work is done, to insure that any infectious material that is dropped from pipette is absorbed by the cover and not leaked into the cabinet or work surface

19 C. HANDLING SHARP MATERIALS AND NEEDLES Sharp materials and needles can pose a risk of percutaneous exposure or accidental inoculation. To avoid risk of injection, whenever possible work with glassware should be avoided and replaced by use of plastic ware (ex. Replace glass Pasteur pipettes with plastic ones). Chipped or cracked glass should never be used and should be discarded appropriately. Needles and syringes should not be used instead of pipettes, and should never be recapped. They should be discarded in appropriately labeled sharp puncture proof containers. D. PROPER USE OF BIOLOGICAL SAFETY CABINETS - Standard operating procedures of the biological safety cabinet should be well defined and applied by any laboratory personnel planning to work with the BSC. - Cabinets must be validated before being operated. Recertification should take place at regular intervals and should be carried out by certified biosafety professionals. - The cabinet provides safety against aerosol release, but it is the responsibility of the worker to ensure that work is being done with care as to avoid spillage and breakage. - The glass front opening should not be opened beyond the limit level indicated on the hood while in operation. Air circulation openings should not be blocked by work products. - The work surface inside the cabinet should be divided into three sections. A clean area, a working a rea and a disposal area. - Bunsen burners should not be operated inside the cabinet, the heat generated by the burner will cause the flow of air to be disturbed and can distort the safety provided by the BSC. - Unnecessary movements (such introducing hands repeatedly into cabinet, or traffic behind operator) should be kept to a minimum when operating the BSC, as it can disturb the airflow of the cabinet. - The cabinet surface should be wiped with disinfectant before and after usage. E. STANDARD MICROBIOLOGICAL PRACTICES - It is the duty of the laboratory director to reinforce the policy that only concerned personnel can access the laboratory, and to ensure that institutional policies are applied. - The handwashing rule must be applied by all laboratory workers. Handwashing must be done after removing gloves and after handling infectious biological materials and before leaving the laboratory. - Laboratory fridges and cabinets should never be used for storing food. - It should be prohibited to smoke, drink, eat, apply cosmetics or handle contact lenses in the laboratory. - Mouth pipetting should be strictly prohibited. - Needles must not be reused, recapped, bent or broken. They should be properly disposed in appropriate sharps container. Broken glassware should not be used and should be discarded immediately in sharp containers

20 - Workers should work cautiously to minimize aerosol and splash production. - Laboratory work surfaces should be decontaminated daily after work, and whenever needed (for example, in case of spills or splashes) throughout the working day. Decontamination should be done with use of an suitable disinfectant. - All infectious materials and cultures should be decontaminated before disposal. - Training regarding laboratory and biosafety procedures should be given for all laboratory personnel by the laboratory director. Annual procedural updates should be distributed to all laboratory workers. Health surveillance should be carried out regularly. F. PREVENTING THE SPREAD OF BLOODBORNE PATHOGENS Bloodborne pathogens of primary concern are hepatitis B, hepatitis C and HIV. They can be encountered by: - Direct contact; e.g., blood splashing in eye. - Indirect contact; e.g., touching an object contaminated with blood of infected person. - Droplet; inhales droplet from infected person To prevent infection; - Avoid contact with blood and other body fluids - Use breathing barriers such as respiratory masks - Wear disposable gloves, a mask, eyewear, gown (assuming blood and other body fluids can splash) - Cover cuts, sores, remove jewelry including rings before wearing gloves - Change gloves when working on a different subject - After completion of work, remove gloves without contacting the exposed part and dispose in a proper container. - Wash hands and other exposed areas. To reduce risk of exposure; - Use biohazard bags to dispose contaminated material - Use sharps disposable containers - Clean and disinfect all equipment and benches. Bleach disinfectants should be 10% chlorine bleach minimum for use on non-porous surfaces. - Scrub boots, belts with soap and hot water. What to do if you are exposed - Wash needle stick injuries and cuts with soap and water. - If splash occurs around the eyes; irrigate with copious amounts water or sterile normal saline solution for 20 minutes. - Record the incident - Seek medical attention - Hepatitis B active and/or passive vaccination might be needed