Biosafety in the TB Laboratory

Biosafety in the TB Laboratory
Whether you work in the laboratory or just passing through you will feel more confident if you better understand the biosafety practices required to work with the tubercle bacilli. Presented by Peggy Coulter with (Patient Safety Monitoring in International Laboratories) Prepared for the ACTG Conference 24 June 2009

Purpose of Training GCLP Standards: DAIDS supported clinical trials and studies involving human subjects must ensure compliance with federal regulations including procedures to protect the safety of all participants. Safety of laboratory employees must be a top priority for all lab facilities. Purpose of training: The primary focus of SMILE is to monitor laboratory testing of analytes identified for patient safety. This training focuses on the safety of laboratory staff who work with the mycobacteria. The risk of tb infection is higher for lab workers when compared to administrative staff or the community. The potential for becoming infected with MDR and the increasing number of persons infected with HIV including lab workers, adds a new dimension and emphasizes the importance of strict adherence to safety precautions in the lab. The potential for unexpected events must be evaluated to eliminate or reduce possible worker exposure to infectious organisms and to prevent release to the environment. Lab personnel should have the information and training they need to safely and confidently work with mycobacteria in the clinical laboratory. Laboratory work lends itself to multi-tasking. The Tb laboratory is not well suited to this work style and should be discouraged for the safety of all staff working in the area. A hurried worker will increase aerosol hazards by working faster or by becoming distracted.

Abbreviations BMBL- Biosafety in Microbiological and Biomedical Laboratories (CDC) LBM- Laboratory Biosafety Manual (WHO) BSC- Biosafety Cabinet LAI- Laboratory Associated Infection TB- for Mycobacteriology testing Like all good programs there will be abbreviations used during this session.

Objectives: At the end of this training you will be able to:
Locate resources for biosafety guidelines; Describe the elements of biosafety; Identify standard and special practices in biosafety; Select and use appropriate biosafety control measures; Conduct an internal risk assessment and self inspection of the laboratory. Objectives: Locate resources for biosafety guidelines; Describe the elements of biosafety; Identify standard and special practices in biosafety; Select and use appropriate biosafety control measures; Conduct an internal risk assessment and self inspection of the laboratory.

Training Topics Principles of Biosafety
Standard Microbiological Practices Special Practices Personal Protective Equipment Containment Equipment Laboratory Facilities Safety Practices Risk Assessment and Self Inspection There are many powerpoint presentations available on the web which cover the required biosafety practices as listed in the CDC and WHO manuals. These can be found through Google. This training session will cover these topics as they apply to TB testing laboratories in detail using examples, photos, pictures and/or demonstrations. You should have received a self inspection checklist when you came in which can be modified for your laboratory. We will have time at the end for questions.

Resources for Principles of Biosafety
Biosafety in Microbiological and Biomedical Laboratories (BMBL) from CDC at Laboratory Biosafety Manual from W.H.O. at Training will focus primarily on these two resources which can be accessed from our website pSMILE.org or from these websites. I recommend that the Microbiology laboratory obtain one or both of these documents, have the administration in addition to the laboratory staff read it and have it readily available in the laboratory. Additional resources are available on pSMILE some of which are in the SMILE drop-in room for preview.

Introduction to Biosafety
All laboratory facilities must follow “Standard or Basic Precautions”. Biosafety guidelines evolved from the microbiological and biomedical community to reduce laboratory associated infections (LIA’s) and to protect the public health and environment. The biosafety practices should be driven by professional judgment based on a risk assessment, rather than by a biosafety level assigned according to the risk group designation of the suspected pathogenic organism. The risks to laboratory workers depend on: the frequency specimens positive for M. tb are processed in the lab, the number of specimens handled by an individual worker, and safety practices and equipment within the laboratory.

Principles of Biosafety
Two basic elements: Containment Risk Assessment Reading the principles of Biosafety can be somewhat confusing as it applies to all biological and biomedical laboratories in both clinical and animal research. As I mentioned with the training topics, this training session will address these principles as they apply to TB testing in the clinical laboratory. These principles are generally described in two basic elements. Containment fundamentals are the safety practices, safety equipment, and Facility safeguards that protect laboratory workers, the environment, and the public from exposure to the infectious microorganisms that are in the laboratory. Containment is the most important element with strict adherence to standard and special microbiological practices and techniques. Risk assessment is a process that enables the appropriate selection of these same practices, equipment and facility safeguards that can prevent laboratory associated infections.

Routes of transmission for LAI’s
Direct skin, eye, or mucosal membrane; Parenteral inoculation by a contaminated sharp or needle; Ingestion of liquid suspension; Inhalation of infective aerosols. Most if not all laboratories are familiar with standard precautions used to protect against the blood born pathogens. We need to protect the laboratory staff from all laboratory associated infections. Before the appropriate practices, equipment and safeguards can be selected to prevent laboratory associated infections, we need to understand how they are transmitted. These are the known routes of transmission for LAIs. Exposure to laboratory-generated aerosols created while performing routine procedures is the most serious of the hazards encountered by lab personnel because they are widespread in laboratory procedures and are usually undetected, placing the laboratory worker and the other persons in the laboratory at risk of infection. Procedures and equipment that generate respirable size particles also generate larger size droplets that can contain multiple copies of an infectious agent. The larger size droplets settle out of the air rapidly, contaminating the gloved hands and work surface and possible the mucus membranes of staff in the area of the procedure. The potential risk from exposure to droplet contamination requires as much attention to risk assessment as the respirable component of aerosols.

Risk Assessment Pathogenicity of the microorganism
Prevalence of tuberculosis and rate of MDR Types of testing performed or referred Volume of tests Personnel expertise and attitude Facilities and equipment To perform a risk assessment information must be gathered. Understand the pathogenicity of the microorganism Know the prevalence of tuberculosis and rate of MDR Types of testing performed or referred- smear, culture, identification Volume of tests - directly affect staff proficiency in both technical procedures and biosafety practices. Laboratories that receive fewer than 20 specimens per week to process for isolating, identifying, and DST are unlikely to maintain proficiency in the required procedures. 20 processed specimens per week may only produce an average of one M. tuberculosis isolation per week. If requests fall below this level, the laboratory may decide that specimens should be sent to a laboratory that processes a larger number of specimens. Personnel expertise and attitude – Staff can get too confident with experience and shortcuts develop. A careless attitude can be dangerous. Just because it is in the local environment is no excuse to be lax in safety measures. Comment- “I have worked in the TB lab for over 20 years and haven’t caught it yet so it can’t be that infectious”. This tech should be monitored for the safety of others in the laboratory. Facilities and equipment – what is available, what is the condition, maintenance and training records should be reviewed.

Risk Microorganism WHO Risk Group
1 No or low individual and community risk. A microorganism that is unlikely to cause human or animal disease. 2 Moderate individual risk, low community risk. A pathogen that can cause human or animal disease but is unlikely to be a serious hazard to laboratory workers, the community, livestock or the environment. Laboratory exposures may cause serious infection but effective treatment and preventative measures are available and the risk of spread of infection is limited. 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 preventative measures are available. 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 preventative measures are not usually available. The principle hazardous characteristics of a microoganism are: its ability to infect and cause disease in a susceptible human or animal host, its virulence measured by the severity of disease, and the availability of preventative measures and effective treatments for the disease. Both WHO and the BMBL address the risk of infection by microorganisms to both the laboratory worker and the community. This classification is described as Risk Groups. These correlate with but do not equate to biosafety levels. For the purpose of this training, we will be focused on WHO Risk Group 3 which contains Mycobacteria tuberculosis.

Levels of Biosafety, and Testing Levels
Biosafety Level (BSL) Activity One 2 Collect clinical specimens Transport specimens to a higher level testing laboratory May prepare and examine smears of killed tubercle bacilli Two 3 Level One activities Process specimens for microscopy and culture Identify M. tuberculosis Perform DST on M. tuberculosis Three Level One and Two activities Identify all Mycobacterium species from clinical specimens Perform DST against all mycobacteria Conduct research and provide training to other laboratories Biosafety levels consist of combinations of lab practices and techniques, safety equipment, and lab facilities. There are several numbers used and it can be confusing. So to summarize, the TB microorganism is considered a Risk Group 3 microorganism. A testing level of two is for a laboratory which performs smear, culture, identification and/or drug susceptibility for MTb. A biosafety level 3 is recommended for this testing. Biosafety level 3 practices can and must be used in absence of a BSL-3 facility. It is incorrect to assume that all laboratories who perform this testing have a BSL-3 facility.

Basic Microbiology Practices
Policies and access Safety practices Decontamination and Waste Training The biosafety guidelines describe the responsibilities of both administration and the laboratory staff in the basic microbiology practices. It is the responsibility of the organizational and Laboratory Directors to evaluate and ensure the effectiveness of their Biosafety programs, the proficiency of their workers, the capability of equipment, facilities and management practices to provide containment of these microorganisms. Policies and access: The Laboratory supervisor must enforce institutional policies that control access to the laboratory. A sign incorporating the universal biohazard symbol must be posted at the entrance to the laboratory when infectious agents are present. An effective integrated pest management program is required. All lab personnel and particularly women of child-bearing age should be provided with information regarding immune competence and conditions that may predispose them to infection. Individuals having these conditions should be encouraged to self-identify to the institution’s healthcare provider for appropriate counseling and guidance. Many of the safety practices are the same as standard precautions: Persons must wash their hands after working and before leaving the laboratory. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food must not be permitted in laboratory areas. Mouth pipetting is prohibited. A sharps policy must be developed and implemented. Plasticware should be substituted for glassware whenever possible. All procedures should be performed to minimize the creation of splashes and/or aerosols. Specific procedures include: Collecting sputum, Adding decontamination solutions, Working with bacteriological loops, Pipetting, Centrifugation, Mixing, vortexing, Pouring into the disinfectant. Decontamination and Waste: Work surfaces must be decontaminated after work is complete and after any spill or splash of potentially infectious material with an appropriate disinfectant. All cultures, stocks, and other potentially infectious materials must be decontaminated before disposal using an effective method (e.g., autoclave, chemical disinfection, incineration, or other validated decontamination method). A method for decontaminating all laboratory wastes should be available in the facility, preferably within the laboratory. Materials to be decontaminated outside of the immediate laboratory must be placed in a durable, leak proof container, secured for transport, and packed in accordance with applicable local regulations. Training: Laboratory supervisors are responsible for educating all laboratory personnel in the concepts of bio-safety and for ensuring that safety procedures are followed. Techniques can significantly impact aerosol output and dose. The worker who is careful and proficient will minimize the generation of aerosols. When a new procedure is introduced, each step of the operation should be evaluated for potential biohazards. Personnel must receive annual updates and additional training when procedural or policy changes occur.

Posted information must include the laboratory’s biosafety level, the supervisor’s name (or other responsible personnel), telephone number, and required procedures for entering and exiting the laboratory. Agent information should be posted in accordance with the institutional policy.

Specimens Specimen containers
Specimen containers may be of glass but plastic is preferred. They must be strong and not leak when the cap or stopper is correctly applied. Containers must be correctly labeled to facilitate identification. Specimen requisition forms must not be wrapped around the containers but placed in separate, preferably waterproof envelopes or bags. Specimens must be collected in clean containers, free from paraffin and other waxes or oils. The following specifications are recommended: • Wide-mouthed (at least 35mm in diameter) so that the patient can expectorate easily inside the container without contaminating the outside; • Volume capacity of 50ml; • Made of translucent material in order to observe specimen volume and quality without opening the container; • Made of single-use combustible material to facilitate disposal; • Screw-capped to obtain a water-tight seal to reduce the risk of leakage during transport; • Easily-labeled walls that will allow permanent identification; An alternative container: The 28ml Universal bottle, which is a heavy glass, screw-capped bottle. This container is reusable after thorough cleaning and sterilization in boiling water for at least 30 minutes.

Photo of washroom This is a washroom of a facility that uses these glass bottles. There are several steps involved in the required cleaning, decontaminating and sterilization required for their use.

Handling of Specimens Collection Transportation
Receipt of incoming specimens Opening packages Handling of specimens Collection of appropriate specimens and prompt transport of those specimens to the laboratory are critical first steps in the laboratory evaluation of the tb patient. Improper collection, transport and handling of specimens in the laboratory carry a risk of infection to the personnel involved. Standard precautions must always be followed, and appropriate barriers such as gloves, gowns, and eye protection utilized whenever samples are obtained from patients. The specimen should be collected under the direction of a trained health care professional. Collecting a good sputum specimen requires that the patient be given clear instructions and informed of the possibly infectious nature of their secretions. Patients should produce specimens either outside in the open air or away from other people and not in confined spaces such as toilets. The specimen must not contaminate the outside of the tube or collection container and the container must be tightly closed after collection. Shipping the specimens within or between facilities require secondary containers constructed of metal or plastic, which are autoclavable or resistant to the chemical disinfectants, and preferably has a gasket seal. They must be regularly decontaminated. To avoid accidental leakage or spillage, secondary containers or boxes, should be fitted with racks so that the specimen containers remain upright. Standard microbiological practices and procedures must be the minimum requirement for handling of patient specimens suspected of containing M. tb. Personnel who receive and unpack specimens should be aware of the potential health hazards involved, and should be trained to adopt standard precautions, particularly when dealing with broken or leaking containers. Disinfectants must be available in this area. Receipt of incoming specimens (Shipping containers) A separate counter area should be used for specimen receipt and the following applied: • Disposable gloves worn during receipt and inspection of incoming specimens. • The delivery box must be examined for signs of leakage. If leakage is evident the box must be decontaminated or discarded by autoclaving or burning. • The outside of the delivery box must be disinfected using cotton wool or paper towels saturated with a suitable disinfectant (e.g. 5% bleach). Opening packages (Secondary and Primary containers) Open the delivery box carefully and check for cracked or broken specimen containers. Ideally these should be decontaminated without processing and another specimen requested. Check that specimens have been adequately labeled with individual identification numbers and that these correspond with the numbers on the packing list and/or requisitions. Disinfect the inside of the delivery box, discard gloves and wash hands after handling specimen containers. Primary specimen containers should be opened in a biological safety cabinet.

Special Practices enhance worker safety, provide environmental protection and address the risk of handling agents requiring increasing levels of containment. Reminder: BSL-3 practices should be used whenever M.tuberculosis is handled even if the physical facilities are a BSL-2. Working with Mycobacteria tuberculosis necessitates safety practices beyond the basic microbiological practices. We will cover each of these special practices individually. Special Practices enhance worker safety, provide environmental protection and address the risk of handling agents requiring increasing levels of containment. Reminder: BSL-3 practices should be used whenever M.tuberculosis is handled even if the physical facilities are a BSL-2.

All persons entering the laboratory must be advised of the potential hazards and meet specific entry/exit requirements. Animals and plants not associated with the work being performed must not be permitted in the laboratory. All persons entering the laboratory must be advised of the potential hazards and meet specific entry/exit requirements. Having all staff and visitors read through your bio-safety manual followed by a short multiple choice quiz is one suggestion for entry requirements. Animals and plants not associated with the work being performed must not be permitted in the laboratory. An animal can not be easily decontaminated and plants will attract bugs and may provide an unwanted source of mold to the lab.

Laboratory personnel must be provided medical surveillance and offered appropriate immunizations for agents handled or potentially present in the laboratory. Laboratory personnel must be provided medical surveillance and offered appropriate immunizations for agents handled or potentially present in the laboratory. Surveillance by first providing a Tuberculin Skin Test; monitoring the conversion rate of personnel; an initial medical evaluation and if necessary, chest x-ray for new employees; an annual screen, evaluation and counseling, if necessary, for all employees.

A laboratory-specific biosafety manual must be prepared, adopted as policy and made available and accessible to the laboratory staff. This is a written plan that defines safe lab practices, spill and emergency procedures The written plan should include a requirement for employees to review the Biosafety plan annually; Follow the written Chemical Hygiene plan with safe lab practices; Require safety training before working in the TB lab; Describe the use of an annually certified BSC for all aerosol possible producing procedures; Describe PPE provided and required decontamination before leaving the lab. Limit access to the TB lab.

The laboratory supervisor must ensure that the laboratory personnel demonstrate proficiency in standard and special microbiological practices before working in the mycobacteriology lab. The laboratory supervisors are responsible for educating all laboratory personnel in the concepts of bio-safety and for ensuring that safety procedures are followed. When a new procedure is introduced, each step of the operation should be evaluated for potential biohazards. Personnel must receive annual updates or addition training when procedural or policy changes occur. Techniques can significantly impact aerosol output and dose. The worker who is careful and proficient will minimize the generation of aerosols.

Personal Protective Equipment (PPE)
Gowns, lab coats Gloves Respirators, masks, goggles, glasses Shoe cover, boots The basic principle of personal protection is the consistent use of appropriate personal protective equipment while manipulating materials that might contain infectious tubercle bacilli. Employees must be trained on how to properly put on and remove their personal protective equipment. Inadequate training in the proper use of PPE may reduce its effectiveness, provide a false sense of security and could increase the risk to the laboratory worker. (e.g., a respirator could impart more harm than not using one) Outer clothing should be properly hung away from the work area and open flames and not on compressed gas cylinders or fire extinguishers. Glass partitions in doors should not be obstructed by hanging clothing over the glass. Contaminated and uncontaminated garments must be stored separately. Contaminated laboratory coats and gowns should be placed and transported in appropriately marked bags that prevent leakage and must be washed at adequate temperature and time intervals to ensure decontamination.

Lab coats vs. Gowns A lab coat like this is acceptable for work in the general microbiology laboratory. Front buttoned standard laboratory coats alone are unsuitable, as are sleeves that do not fully cover the forearms for working in the TB lab. Additional protection from spills of possible infectious materials is required. An appropriate gown must be selected, with a solid-front such as a tie-back or wraparound gowns, scrub suits, or coveralls. These must have ribbed cuffs and be made of fluid impermeable material. This wrap around can be worn over a lab coat or alone. A back tied gown must be worn over a lab coat. Gowns must be fastened completely, cuffs not rolled or pushed up the arm. This is worn to protect skin and to prevent contamination of clothes during procedures that are likely to generate splashes of potentially infectious material. Clothing must be changed when contaminated and, soiled or not, these must be removed before leaving the laboratory. Reusable clothing is decontaminated with appropriate disinfectant before being laundered.

Approved Type Correct Size Donning Proper Use Removal
Gloves Approved Type Correct Size Donning Proper Use Removal Gloves must be worn to protect hands from exposure to hazardous materials. Gloves must be approved for use as a medical glove. Food service or household gloves are not appropriate for work at the bench. General-purpose utility gloves (e.g., rubber household gloves) for housekeeping chores involving potential blood contact and for device cleaning and decontamination procedures can be used but attention must be given to their correct washing, removal, cleaning and disinfection and must be discarded if they are pealing, cracked or discolored, or if they have punctures, tears, or other evidence of deterioration. Disposable non-sterile latex, nitrile, and vinyl gloves provide adequate barrier protection. Powdered latex gloves are a risk factor for the induction of latex allergic reactions in healthcare workers due to occupational exposure. As allergies to latex are developing in the healthcare workforce, alternatives to latex gloves must be available. Gloves must be available in several sizes at the workstation. Gloves which are too small may tear. Gloves too large may leave gaps at the cuff exposing the worker to contamination, may affect the technical abilities of the worker, and may result in unsafe practices. The workers hands may change sizes (e.g. swelling due to heat or personal conditions). Laboratory workers must be trained to practice aseptic technique when putting gloves on and when removing them. Gloves must be pulled over the knitted wrists of the gown rather than worn inside to prevent the worker’s skin from exposure. Disposable gloves should be examined for visible defects after donning and before commencing work. Less skin contamination may be possible when wearing two pairs of disposable gloves than when using a single pair, due to protection during gloves changes from contamination at the bench. This is not a required practice and may be uncomfortable and hard to perfect. Disposable gloves must not be washed and reused, since this degrades the protective function of the glove. Gloves must be changed when contaminated, integrity has been compromised, or when otherwise necessary. Gloves do not need to be changed during routine laboratory activities that result in contaminating the gloves (e.g., wiping the probe of an automated hematology analyzer). Rather gloves, should be changed when these tasks are completed. Laboratory workers must be diligent in avoiding contamination of a clean area by contact with contaminated gloves. Gloves must be worn at the specimen receiving and setup areas and in the TB/virology laboratories, and when hands may contact potentially infectious material, contaminated surfaces, or equipment. Gloves must be removed and hands washed prior to leaving the laboratory. Proper techniques will protect the worker from skin contamination from contaminated gloves. Demonstrate both correct and incorrect methods. Gloves must not be worn outside the laboratory. Hand washing protocols must be rigorously followed. Dispose of used gloves with other contaminated laboratory waste. Gloves must be removed before handling telephones, uncontaminated laboratory equipment, doorknobs etc. Alternatively, specific devices, such as computer keyboards may be specially labeled as a biohazard and used only with gloved hands. Some laboratories have phones designated for use with a glove, speaker phones should be made available in the TB laboratory as a dirty gloved hand should not hold a phone next to the face.

Open-toed footwear is not appropriate in the laboratory.
Shoes and Covers Open-toed footwear is not appropriate in the laboratory. Due to different cultural lifestyles and dress this will be hard to enforce but both the CDC and WHO state that open toed footwear must not be worn in the laboratory. Fluid impermeable footwear is recommended. Shoes should be comfortable, rubber-soled, and cover the entire foot. Disposable, fluid-resistant shoe covers can be worn where splashing is expected. If a large spill occurs and there is the potential of contaminating the worker’s shoes, water-impermeable shoe covers should be worn.

Respirator program implemented by the laboratory's safety officer or person designated to perform this task and should include written procedures concerning how to: select the appropriate respirator, conduct fit-testing, and train personnel on the use, fit checking, and storage of the respirator. Respirators must be used only by those employees who are physically able to breathe through a respiratory device and only in accordance with instructions and training received. Untrained or physically exempt employees should not work in respiratory protection areas. Respirator protection must accommodate facial hair and/or personal glasses. Respiratory protective devices must be NIOSH- certified N-series filter with 95% efficiency (N-95) appropriate for use. When respiratory protection devices are required, a written respiratory protection program must be established and maintained. This program includes standard operating procedures covering hazard evaluations; medical evaluation and user surveillance to include: Selection of appropriate respirators How to conduct fit testing Fit testing to obtain face seal leakage of no more than 10% Fit different facial sizes and characteristics (make available in different styles and sizes) Check for fit with each use Training of personnel in use, fit, checking and storage of respirator.

Correct Type and Fit of Respirators
Dust Surgical Mask Dust and Paint Fumes All masks and respirators are not equal. Surgical masks are not NIOSH certified respirators and must not be worn to provide respiratory protection. Masks for dust and or paint fumes are not appropriate. A fancy mask does not make it better. Show different types of N-95 masks and non N-95 masks. Like gloves, several types and sizes should be available in the laboratory for different facial types depending on the number of persons and visitors permitted in the TB laboratory. These may be stored outside the laboratory. N-95

PAPR Beards prevent proper use of respiratory devices. Persons with beards cannot be fit tested due to the inability to get a good facial seal. OSHA law prohibits those individuals to wear a mask until the person is clean-shaven and fit-tested. Individuals, like Kurt shown here, can not be fitted with a respirator and should use a powered air purifying respirator (PAPRs) in the TB laboratory. Demonstrate the correct procedure for putting on a respirator. Open the respirator by pushing slightly on both sides. Shape the nose-bridge. Place the respirator under the chin with the chin-flap fully open. Holding the respirator against the face, place the lower head-strap around the neck below the ears and the top head-strap above the ears on the crown. This is how the respirator should appear when correctly donned. Mold the nose area to the shape of the face by pinching the nose-bridge at the top and sliding down to the bottom of the bridge. Fit-check the face-seal as follows: Place both hands over the respirator without disturbing its position, exhale sharply, and if you detect air-leaks, re-adjust the respirator, if re-adjustment does not fix the air leak, ask for a different size of respirator. Inadequate training in the proper use of PPE may reduce its effectiveness, provide a false sense of security, and could increase the risk to the laboratory worker. For example, a respirator may impart a risk to the wearer independent of the agents being manipulated. The respirator may be reused but should be replaced at regular intervals depending on length of time in use. This should be covered in the Respiratory Program. The respirator may be stored individually inside a plastic Ziploc baggie, or equivalent (large manila envelope) to keep the inside from contamination. One labeling method is to sign your name with a Sharpie permanent marker somewhere on the non-latex straps so that these are not mixed up between persons. The respirator must be replaced when: The respirator becomes physically damaged The integrity of the respirator is impaired Irritation occurs. On a regular basis as decided by the health officer or laboratory supervisor. Dispose of the respirator in the regular trash receptacle. Respirators must not be worn outside the laboratory areas.

Safety Equipment Needle locking syringes Centrifuge safety carriers
Microburners Biosafety Cabinets (BSC) The use of proper safety equipment provides primary personnel protection. For example, needle locking syringes, micro-burners, safety centrifuges cups and sealed rotors are among the engineered devices that protect laboratory workers from biological hazards. Safety equipment that is not used consistently or properly is hazardous, especially when the user is unaware of the malfunction. Training in the correct use of equipment, proper procedure, routine inspections and potential malfunctions, and periodic re-certification of equipment, as needed, is essential.

Centrifuge Safety Satisfactory mechanical performance is required for safety in the use of laboratory centrifuges. Infectious airborne particles may be ejected when centrifuges are used. However, good centrifuge technique and securely capped tubes offer adequate protection against infectious aerosols and dispersed particles. Centrifuges must be operated according to the manufacturer’s instructions. Centrifuges must be placed at such a level that workers can see into the bowl to place buckets correctly. (Photo shows centrifuge too high up and back for a person of short stature.) Centrifuge tubes and specimen containers for use in the centrifuge should be made of thick-walled glass or preferably of plastic and should be inspected for defects before use. Glass tubes may break under the stress of centrifugation. If a centrifuge tube breaks, the liquid will splash or be blown out and aerosolized. Tubes and specimen containers should always be securely capped before centrifuging. Sealable centrifuge buckets (safety containment cups) designed to contain aerosols that will be generated if a tube leaks or breaks must be used. These cups must be loaded, equilibrated, sealed and opened in a biological safety cabinet. Screw top centrifuge tubes should be used for potentially infectious material. Buckets should be correctly balanced. When using fixed angle-head centrifuge rotors, care must be taken to ensure that the tube is not overloaded as it might leak. The amount of space left between the level of the fluid and the rim of the centrifuge tube should be given in manufacturer’s instructions. The centrifuge head must be balanced while in use. An out-of-balance head vibrates and may break. As an added safety precaution, matched tubes should contain 70% ethanol rather than water, which may limit the risk of infection should breakage occur. Distilled water or alcohol (propanol, 70%) may be used for balancing empty buckets. Saline or hypochlorite solutions should not be used as they corrode metals. O-rings on the centrifuge caps must be examined daily to assure that the seal is intact and that the integrity of the unit is maintained; cracked or otherwise faulty O-rings must be replaced before equipment is reused. Centrifuge rotors and buckets should be inspected daily for signs of corrosion and for hair-line cracks. The interior of the centrifuge bowl should be inspected daily for staining or soiling at the level of the rotor. If staining or soiling are evident then the centrifugation protocols should be re-evaluated. Buckets, rotors and centrifuge bowls should be decontaminated after each use. After use, buckets should be stored in an inverted position to drain the fluid. Centrifuges should preferably be fitted with an electrically operated safety catch which prevents the lid from being opened while the rotor is spinning. Do not touch any centrifuge head while it is spinning. Touching it may not only cause injury, it may also cause rapid or erratic stops which stir and resuspend the sediment. Some centrifuges are equipped with a brake to gradually slow the spinning head.

Biosafety Cabinets (BSC)
All procedures involving the manipulation of infectious materials must be conducted within a BSC, or other physical containment devices. No work with open vessels is conducted on the bench. When a procedure cannot be performed within a BSC, a combination of personal protective devices, such as centrifuge safety cup with sealed rotor, must be used. The most important piece of containment equipment is a well maintained and properly functioning biological safety cabinet in which manipulations of infectious microorganisms are performed. Because of the potential for aerosol generation, specimen containers must be opened and direct smears prepared and air dried in a BSC. Smears may be dried and heat-fixed by placing the slide on a warmer in the BSC and heating. Aerosol-generating procedures such as blending, mixing, pipetting, inoculation of media, and sonication must be performed in a BSC. Sputum specimen containers must be opened, chemicals for digestion added, and the processed specimen placed in appropriate centrifuge tubes in a BSC. Activities such as inoculation of both liquid and solid medium for primary isolation, identification of all Mycobacterium species using rapid methods, and susceptibility testing of M. tuberculosis must be done in the BSC. Not all procedures in the TB laboratory need to be performed in a BSC. Heat-fixed smears may contain viable tubercle bacilli, but they are not easily aerosolized if dried on a slide. Personnel may remove fixed slides from the BSC and stain them without wearing a respirator or using a BSC. Stain reagents for both light and fluorescence microscopy contain phenol, which kills tubercle bacilli during the staining process. When tubercle bacilli are inoculated onto a solid medium contained in a test tube, the screw cap is left loose for up to one week to allow water vapor, oxygen, and carbon dioxide to diffuse. Droplet nuclei do not form in the undisturbed tube. Therefore, examining closed culture vessels (e.g., slant tubes, sealed agar plates) may be done on an open bench. All cultures of specimens must be assumed to contain M. tuberculosis until tests prove otherwise, and specimens from patients having mixed infections with two Mycobacterium species can occur. Any manipulations involving open tubes must be done in the BSC.

Use of BSC A BSC is the most important piece of containment equipment but only if properly installed, appropriate air velocity is maintained during use, proper procedures are used. A properly certified and operational BSC is an effective engineering control which must be used in concert with the appropriate practices, procedures and other administrative controls to further reduce the risk of exposure to potentially infectious microorganisms. Maximum containment potential is achieved only through strict adherence to proper practices and procedures. If the air curtain is disrupted (e.g., movement of materials in and out of a cabinet, rapid or sweeping movement of the arms) the potential for contaminant release into the laboratory work environment is increased as is the risk of product contamination. The use and limitations of biological safety cabinets should be explained to all potential users, with reference to national standards and relevant literature. Written protocols or safety or operations manuals should be issued to staff. In particular, it must be made clear that the cabinet will not protect the operator from spillage, breakage or poor technique. The cabinet must not be used unless it is working properly. The glass viewing panel must not be opened when the cabinet is in use. Apparatus and materials in the cabinet must be kept to a minimum. Air circulation at the rear plenum must not be blocked. Bunsen burners must not be used in the cabinet. The heat produced will distort the airflow and may damage the filters. An electric microincinerator is permissible but sterile disposable transfer loops are better. Other personnel activities in the room (e.g., rapid movements near the face of the cabinet, walking traffic, room fans, open/closing room doors, etc.) may also disrupt the cabinet air barrier. Extra supplies (e.g., additional gloves, culture plates or flasks, culture media) should be stored outside the cabinet. Only the materials and equipment required for the immediate work should be placed in the BSC. UV lights are not required in the cabinet but if used: Must be cleaned weekly with alcohol soaked gauze to remove dust and dirt that block the germicidal effectiveness. UV light has little penetrating power and is easily blocked by grease. Checked weekly with UV meter for appropriate intensity or checked every 3 months for effectiveness. Turned on for a minimum of one hour after work is complete. Turned off when room is occupied to protect skin and eyes from exposure Close sash when on.

Use of biological safety cabinets
Demonstrate sitting at the BSC. Technologists should be trained in proper operation and cleaning procedures. The work surface must be organized so that air circulation is not compromised. A written checklist should be prepared for all materials necessary for the activity and necessary materials placed in the BSC before beginning work. Paperwork should never be placed inside biological safety cabinets as it can block grills and will affect airflow. Materials or equipment placed inside the cabinet may cause disruption of the airflow. Aerosol generating equipment should be placed toward the rear of the cabinet. Bulky items such as biohazard bags, discard pipette trays should be placed to one side of the interior of the cabinet. The remaining materials should be placed as far back as possible in the cabinet, toward the rear edge of the work surface and at least four inches away from the front grille of the cabinet. The surfaces of all materials and containers placed into the cabinet should be wiped with 70% ETOH to reduce the introduction of contaminants to the cabinet environment. Plastic-backed absorbent toweling can be placed on the work surface but must not cover the front or rear grille openings or obstruct them in any way. This toweling should be soaked with the disinfectant to facilitate routine cleanup and reduce splatter and aerosol formation in case of a spill. If the cabinet has been shut down, the blowers should be operated at least four minutes before beginning work to allow the cabinet to “purge” to remove any suspended particulates in the cabinet. Before beginning work, the worker should adjust the stool height so that his/her face is above the opening. Traffic behind the operator should be minimized. If there is a drain valve under the work surface, it should be closed prior to beginning work in the BSC. All work must be delayed for approximately one minute after placing the hands/arms inside the cabinet. This allows the cabinet to stabilize, to “air sweep” the hands and arms, and to allow time for turbulence reduction. A sweeping motion of the arms will disturb the airflow. Moving arms in and out slowly, perpendicular to the face opening of the cabinet will reduce this risk. Resting arms on the front grille, will block the grill opening, particles from the room air may flow directly into the work area, rather than being drawn down through the front grill. Raising arms slightly will alleviate this problem. The front and other air grills must not be blocked with toweling, notes, discarded plastic wrappers, pipeting devices, etc., as this will disrupt the airflow causing potential contamination of the material and exposure of the operator. All operations should be performed at least four inches in from the front grille, carried out in the middle of the working surface and be visible through the viewing panel. Active work should flow from clean to contaminated area across the work surface. When work is complete, the toweling can be folded with the absorbent side inward and disposed as biohazardous waste. When work is complete, with the blower running, all containers and equipment should be surface decontaminated and removed. The work surface, interior walls and the interior surface of the window should be wiped with a 1:100 dilution of household bleach, or other disinfectant as determined to be effective may be used. When bleach is used, a second wiping with sterile water or the 70% alcohol is needed to remove the residual chlorine, which may eventually corrode stainless steel surfaces. Close the BSC and leave the blower on for at least 5 minutes. Remove gloves carefully inside out, remove gown, wash hands then turn off blower or leave as per laboratory procedure. Decontamination of biological safety cabinets prior to service by an engineer should be performed following manufacturer’s recommendations.

Facilities Ventilation Temperature control Sinks, eyewash, trashcans
Furniture and decontamination The lab facility must be designed in such a manner that the safety of the employees and the quality of the work is not compromised. Contrary to common belief containment laboratories need not be overly sophisticated and expensive. Some quick comments about facility safeguards: Ventilation Windows must be closed while work is in process. Sophisticated and expensive air conditioning is not an essential requirement, but during working hours, air must be continuously extracted to the outside of the laboratory either through a biological safety cabinet or through extraction fans in walls or windows. Air currents must not pass over the area of smear preparation in the direction of the lab worker preparing the smears. Ventilation standards should be considered in relation to the number of specimens processed per year and the prevalence of tuberculosis among these specimens. Temperature control As per good clinical lab practice, room temperature of the TB laboratory must be recorded and maintained. While an air conditioner is not required, a room that is too warm may result with employees removing PPE, not fastening it completely or rolling up sleeves. Sinks, eyewash, trashcans Sinks should be located within 100 feet of hazardous chemicals. Hand washing sinks should be separate from sinks used for disposal of blood and body fluids and should be located near the exits. Alcohol-based foam dispensers can be placed in convenient locations where access to hand washing sinks is limited; however the use of such foams does not preclude the use of soap and water as soon as feasible. Eyewash stations should be conveniently located where ever hazardous materials are located. These devices should be tested each week to ensure proper functioning and to flush out stagnant water. Portable eyewash systems must be designed and monitored to ensure the biological and chemical purity is maintained. Adequate and conveniently located biohazard containers for disposal of contaminated materials must be provided. Foot operated containers are recommended to prevent recontamination of the hands after washing. Adequate decontaminating containers for reusable supplies must be provided. Furniture and decontamination Furniture must be easy to clean and decontaminate, so it must be made of wood, metal or other fluid impermeable material. Counters must be able to hold the weight.

Door fitted with a filter.

Plan of a culture laboratory
TB testing should always be performed in laboratories, physically separated from other laboratory areas. The objective is to reduce the infection risk, not only to staff working in the TB lab, but also to others in the same building. The detailed arrangement of a tuberculosis laboratory will vary according to the size and shape of the available room, the type of laboratory activity and whether other work is also done in the same room. Nevertheless, the most important aspect in tb lab design is to ensure a logical flow of specimens and activities, from the least contaminated to the most, and air is then exhausted to the outside without recirculation away from occupied areas and air supply intakes of any building. Air movement within the laboratory can be tested with a simple indicator (e.g., a strip of tissue paper placed in a 1.5-inch by 12-inch slot in the door) or with more complex devices (e.g., magnehelic gauges).

Handling of contaminated laboratory supplies
Glassware Sputum containers Applicator sticks, paper, pipettes Positive and negative slides A container with appropriate disinfectant should be present in the BSC into which used, contaminated pipettes, loops and grinding vessels should be placed. The container should be deep enough to completely cover the discarded items. Used pipettes, wire loops etc. should be soaked for two hours then they can be washed, sterilized and re-used. Close to the BSC there should be containers with lids, for discarded specimen containers and tubes with bacterial suspensions. Contaminated fluids should not be poured down drains but carefully poured into a splashproof containers containing appropriate disinfectant. Glassware should be substituted with plastic whenever possible. Broken glassware should be removed by a brush and dustpan, tongs or forceps and decontaminated in an appropriate disinfectant before disposal. Plastic sputum containers should be disposed of by incineration. Used glass sputum containers can be recycled after autoclaving and thorough washing. Wooden applicators and paper should be disposed of by incineration. Contaminated or used pipettes should be soaked for two hours in a bactericidal solution, washed and sterilized before re-use. Positive and negative slides should be autoclaved to prevent their re-use.

Waste Handling Potentially infectious materials must be placed in a durable, leak proof container during collection, handling, processing, storage, or transport within a facility. No infected material should leave the laboratory except when it is properly packed for transport to another laboratory. No infected material should leave the laboratory except when it is properly packed for transport to another laboratory. All pathological material, smears, cultures and containers should at least be disinfected and preferably sterilized before disposal or re-use. Sterilization means the complete destruction of all organisms, while disinfection implies the destruction of organisms causing disease. Sterilization is usually accomplished by heat and disinfection or by treatment with chemicals.

Cleaning laboratory materials
Many germicidal products claim activity only on precleaned items so precleaning is essential to achieve proper disinfection or sterilization. Precleaning must be carried out with care to avoid exposure to infectious agents. Cleaning is the removal of dirt, organic matter and stains by washing with a soap or detergent. Dirt, soil and organic matter can shield microorganisms and can interfere with the killing action of decontaminants. It is quite common to use the same chemical germicide for precleaning and disinfection.

Disinfection and Sterilization
A basic knowledge of disinfection and sterilization is crucial for biosafety in the laboratory. Laboratory equipment should be routinely decontaminated, as well as, after spills, splashes, or other potential contamination. The purpose of decontamination in the Microbiology Laboratory is to protect the laboratory worker, the environment, anyone who enters the lab or handles lab products removed from the lab. To start, I’ll quickly define a few terms: Decontamination – Any process for removing and/or killing microorganisms. Disinfectant – A chemical or mixture of chemicals used to kill microorganisms, but not necessarily spores. Disinfectants are usually applied to inanimate surfaces or objects. Sterilization is a procedure that kills all living microorganisms and viruses including bacterial endospores. Autoclaving is an example of this method. Read labels: Intermediate-level disinfection kills microorganisms including MTb, all fungi and inactivates most viruses. Corresponds to EPA-approved “hospital disinfectants” that are also “tuberculocidal”. Refer to either the CDC or WHO manuals for more information on disinfectants. Some examples are: Amphyl - used at 5% Sodium hypochlorite solutions, as domestic bleach, contain 50 g/l available chlorine and should therefore be diluted 1:50 or 1:10 to obtain final concentrations of 1 g/l and 5 g/l, respectively Alcohols used at 70% as a rinse due to evaporation before an acceptable contact time would be attained. Low-level disinfection kills most vegetative bacteria except MTb, some fungi and inactivates some viruses; EPA-approved as “hospital disinfectants” or “sanitizers”. Large space decontamination for BSL-3 should use surface decontamination not fumigation. Surface decontamination uses liquid by flooding area for periods up to several hours, not using high-level disinfectant which is reserved for instruments or devices.

Autoclaves Autoclaving is the optimal initial sterilization procedure and staff should be carefully instructed in the correct procedure. This is a topic that would be well served to have its own training session. Ideally, the autoclave should be inside the tuberculosis laboratory to prevent contaminated material from being discarded or washed before decontamination. Autoclaves should be tested periodically using autoclave indicator tape and bacterial spores or an equivalent indicator system to ensure that temperatures and process kills all microorganisms. Waste generated from tuberculosis testing must be autoclaved at a minimum temperature of 121°C for a minimum period of 15 minutes. After autoclaving, waste material may be burned or buried. Re-usable articles may be washed and resterilized.

Boiling and burning Peripheral laboratories may not have autoclaves and an alternative must be provided for the disposal of specimen containers and other items. The simplest methods for treating infected material are boiling and burning. A domestic pressure cooker can be used in much the same way as an autoclave, although its capacity is limited. Alternatively, a boiler adapted from an oil-drum or petrol-can, can be suspended over a wood fire and infectious material boiled for 60 minutes before washing or discarding by burning.

Safety Practices Pipetting Microscopy Handwashing
Use of pipettes and pipetting aids in the TB lab is more restrictive for safe practices A pipetting aid must always be used. Pipetting by mouth must be prohibited. All pipettes should have cotton plugs to reduce contamination of pipetting devices. Air should never be blown through a liquid containing infectious agents. Infectious materials should not be mixed by alternate suction and expulsion through a pipette. Liquids should not be forcibly expelled from pipettes. Mark-to-mark pipettes are preferable to other types as they do not require expulsion of the last drop. Contaminated pipettes should be completely submerged in a suitable disinfectant contained in an unbreakable container. They should be left in the disinfectant for the appropriate length of time before disposal. A discard container for pipettes should be placed within the biological safety cabinet, not outside. Keep an absorbent material placed on the working surface to avoid dispersion of infectious material dropped from a pipette. Films and smears for microscopy- Fixing and staining of blood, sputum and fecal samples for microscopy do not necessarily kill all organisms or viruses on the smears. These items should be handled as sharps, stored appropriately, and decontaminated and/or autoclaved before disposal.

Handwashing Hand washing: Simple and Effective- Frequent hand hygiene is the most important safety precaution in preventing the spread of infection. Hands must be decontaminated to avoid transfer of microorganisms to other surfaces and environments. Gloves must be worn when handling biohazardous materials, however, this does not replace the need for regular and proper hand-washing by laboratory personnel. In most situations, thorough washing of hands with ordinary soap and water is sufficient to decontaminate them, but the use of germicidal soaps is recommended in high-risk situations. Hands should be thoroughly lathered with soap, using friction, for at least 10 s, rinsed in clean water and dried using a clean disposable paper towel (if available, warm-air hand-dryers may be used). A waterless alcohol agent may be used if hands are not visibly soiled, followed by soap and water when available. Hands must be washed frequently during the day as good infection control practice. Hands must be washed after removing gloves, before leaving the laboratory, before and after contact with patients, and before eating, drinking, or the manipulation of contact lenses. Hands should be washed immediately after accidental contact with blood, body fluids, and contaminated materials. Soap products that may disrupt skin integrity should be avoided. Using a hand moisturizer may reduce skin irritation cause by frequent hand washing. Foot- or elbow-operated faucets at the sink are recommended but when these are not available, a paper towel must be used to turn off the faucet handles to avoid re-contaminating washed hands. Disposable paper towels should be available for drying hands.

Staff should be observed in their biosafety practices
Training Initial on hire Annual updates Staff should be observed in their biosafety practices Training - staff should be trained and monitored in use of safety practices for the safety of all lab staff in addition to their family members. Laboratory supervisors are responsible for educating all laboratory personnel in the concepts of biosafety and for ensuring that safety procedures are followed; when a new procedure is introduced, each step of the operation should be evaluated for potential biohazards. Personnel must receive annual updates or addition training when procedural or policy changes occur. Bad habits can become rooted over time so observation is strongly recommended to remind staff of the correct practices. Techniques can significantly impact aerosol output and dose. The worker who is careful and proficient will minimize the generation of aerosols.

Laboratory safety does not just happen.
The best defense against a laboratory accident is a well-thought-out plan to neutralize its effects as quickly and effectively as possible. recognize that accidents can and will occur formulate a plan of action discuss ways to minimize and prevent accidents Laboratory safety does not just happen. recognize that accidents can and will occur the best defense against a laboratory accident is a well-thought-out plan to neutralize its effects as quickly and effectively as possible. formulate a plan of action to neutralize the potential harmful effects of an accident as rapidly and effectively as possible No accident should be considered insignificant; however, assessment of the seriousness of each accident is necessary to determine the most appropriate course of action. Be prepared for an accident by having the following readily accessible in or near areas where accidents are most likely to occur: a supply of paper towels or large cloths a wide-mouthed (to facilitate rapid pouring) container of disinfectant a supply of industrial face masks capable of filtering particle sizes between 1μm and 5μm

Spill Procedures Laboratory accidents in the TB laboratory result in possible formation of aerosols. Spills involving infectious materials must be contained, decontaminated, and cleaned up by staff properly trained and equipped to work with infectious material. Accidents that occur in tuberculosis laboratories may be divided into two types: those that generate limited aerosols and those that produce a large volume of potentially infected aerosols. A limited aerosol may, for example, be created by breaking a single culture tube of egg medium or spilling the contents of a sputum specimen. In these instances, the solid medium and thick mucoid nature of the sputum specimen greatly limit large numbers of tubercle bacilli from being aerosolized. In the event of any spillage within the BSC, the cabinet should not be switched off. For a small spill-within the BSC- remove contaminated paper, place in biohazard bag, or wipe splatter with disinfectant. Continue to run the BSC. Remove gloves and wash hands. Large spills within the BSC- all items must be surface decontaminated and removed from cabinet. Close drain valve, pour solution onto work surface and leave for minutes. Soak up with paper towels and discard. Empty drain pan into disinfectant and flush with water. Spill outside the BSL-3 laboratory- A large volume of potentially infectious aerosols may be generated by breaking one or more tubes of liquid containing a high concentration of tubercle bacilli, eg. bacterial suspensions or unbalanced centrifuge tubes. Wearing a respirator clean up the spill after others have left the area, do not wait for aerosols to settle. General rules: BSCs should be on and centrifuges off. Wear a respirator when cleaning a spill PPD negative personnel should be tested 3-6 months after the accident.

Emergency Procedures Puncture wounds, cuts and abrasions
Ingestion of potentially infectious material Potentially infectious aerosol release (outside a biological safety cabinet) Broken containers and spilled infectious substances Breakage of tubes containing potentially infectious material in centrifuges not having sealable buckets Breakage of tubes inside sealable buckets (safety cups) Puncture wounds, cuts and abrasions The affected individual should remove protective clothing, wash the hands and affected area(s), apply an appropriate skin disinfectant, and seek medical attention as necessary. The cause of the wound and the organisms involved should be reported, and appropriate and complete medical records kept. Ingestion of potentially infectious material Protective clothing should be removed and medical attention sought. Identification of the material ingested and circumstances of the incident should be reported, and appropriate and complete medical records kept. Potentially infectious aerosol release (outside a biological safety cabinet) All persons should immediately vacate the affected area and any exposed persons should be referred for medical advice. The laboratory supervisor must be informed at once. No one should enter the room for an appropriate amount of time (30 min), to allow aerosols to be carried away and heavier particles to settle. If the laboratory does not have a central air exhaust system, entrance should be delayed(e.g. for 24 h). Signs should be posted indicating that entry is forbidden. After the appropriate time, decontamination should proceed, supervised by the laboratory supervisor. Appropriate protective clothing and respiratory protection must be worn. Broken containers and spilled infectious substances Broken containers contaminated with infectious substances and spilled infectious substances should be covered with a cloth or paper towels. Disinfectant should then be poured over these and left for the appropriate amount of time. The cloth or paper towels and the broken material can then be cleared away; glass fragments should be handled with forceps. The contaminated area should then be swabbed with disinfectant. If dustpans are used to clear away the broken material, they should be autoclaved or placed in an effective disinfectant. Cloths, paper towels and swabs used for cleaning up should be placed in a contaminated-waste container. Gloves should be worn for all these procedures. If laboratory forms or other printed or written matter are contaminated, the information should be copied onto another form and the original discarded into the contaminated-waste container. Breakage of tubes containing potentially infectious material in centrifuges not having sealable buckets If a breakage occurs or is suspected while the machine is running, the motor should be switched off and the machine left closed (e.g. for 30 min) to allow settling. If a breakage is discovered after the machine has stopped, the lid should be replaced immediately and left closed (e.g. for 30 min). The laboratory supervisor must be informed. Strong (e.g. thick rubber) gloves, covered if necessary with suitable disposable gloves, should be worn for all subsequent operations. Forceps, or cotton held in the forceps, should be used to retrieve glass debris. All broken tubes, glass fragments, buckets, trunnions and the rotor should be placed in a noncorrosive disinfectant. Unbroken, capped tubes may be placed in disinfectant in a separate container and recovered. The centrifuge bowl should be swabbed with the same disinfectant, and then swabbed again, washed with water and dried. All materials used in the clean-up should be treated as infectious waste. Breakage of tubes inside sealable buckets (safety cups) All sealed centrifuge buckets should be loaded and unloaded in a biological safety cabinet. If breakage is suspected within the safety cup, the safety cap should be loosened and the bucket autoclaved. Alternatively, the safety cup may be chemically disinfected.

Incidents that may result in exposure to infectious materials must be immediately evaluated and treated according to procedures described in the laboratory biosafety manual. All such incidents must be reported to the laboratory supervisor. Medical evaluation, surveillance, and treatment should be provided and appropriate records maintained. Staff employed in tuberculosis laboratories should be selected carefully; they should be physically and mentally capable and should accept the responsibility for using Biosafety practices. Training in laboratory procedures and strict adherence to safety measures should be accompanied by a simple surveillance program whereby the health status of laboratory staff is monitored regularly. Each laboratory worker should have a confidential monitoring file in which screening procedures for tuberculosis as well as other health-related data are recorded. The elements of a disease monitoring program include the following: Pre-employment profiles and baseline screening of laboratory workers A standardized health questionnaire should be completed for each employee. This questionnaire should relate past tuberculosis infection disease, BCG vaccination status, underlying medical conditions which may compromise the susceptibility to tuberculosis and previous contact with confirmed tuberculosis cases. A baseline chest x-ray or a Mantoux tuberculin skin test (TST) should be performed. Strongly positive reactors with skin test diameters of >15mm and symptoms suggestive of tuberculosis should be evaluated clinically and microbiologically. Two sputum specimens, collected on successive days, should be investigated for tuberculosis by microscopy and culture. Confidential HIV testing with pre-and post-test counselling should be offered to all laboratory workers. BCG re-vaccination as a means of preventing tuberculosis in laboratory workers is not recommended. Quarterly monitoring of health status Laboratory workers should declare information on their health status in the form of answers to specific questions relating to the early signs and symptoms of TB. These include: cough for longer than three weeks, weight loss, anorexia, night sweats and the frequent occurrence of colds or other respiratory infection episodes in recent weeks. The laboratory worker’s weight should be recorded during each monitoring exercise and an unexplained loss of more than 10% during the previous quarter should be followed up with clinical and microbiological investigations for tuberculosis. information on health status can be obtained by using a single structured questionnaire Post-exposure monitoring Following an accident in the laboratory the laboratory workers” health monitoring file should be reviewed. S/he should be carefully monitored clinically. Eight weeks after the exposure episode a chest x-ray examination should be performed, together with a TST in cases where the baseline reaction diameter was <10mm.

Support Staff The safe and optimum operation of a laboratory is dependent to a great extent on the support staff, it is essential that such personnel are given appropriate safety training. Engineering and building maintenance services Skilled engineers and craftsmen who maintain and repair the structure, facilities and equipment, must have some knowledge of the nature of the work of the laboratory, and of safety regulations and procedures. Testing of equipment after servicing, e.g. testing the efficiency of biological safety cabinets after new filters have been fitted, may be carried out by or under supervision of the laboratory supervisor. Laboratories or institutions that do not have internal engineering and maintenance services must establish good relationships with local service providers to familiarize them with the equipment and work of the laboratory. Engineering and maintenance staff should only enter laboratories with clearance and supervision by the laboratory supervisor. Cleaning personnel should only enter Mycobcteriology laboratories with clearance and supervision by the laboratory supervisor.

THE MYCOBACTERIOLOGY LABORATORY IN NEED OF IMPROVEMENT
Physical facilities BSL-2 vs BSL-3 It is recognized that some laboratories may not currently meet BSL-3 guidelines because of certain facility limitations, (e.g., not having a complete BSL-3 laboratory). In those laboratories, the laboratory director and supervisor should evaluate the facility, available equipment and work practices to determine what services can be provided without compromising employee health and safety. Policies and procedures must be developed to allow TB testing to follow full BL-3 practices and procedures while working in a BSL-2 laboratory. However, to achieve optimum good laboratory practices the development of a plan to achieve appropriate facility upgrades is needed. When routine work in a BSL-2 facility will be performed using BSL-3 procedures, all work practices should be closely monitored, and all employees should receive tuberculin skin tests at recommended intervals. Some laboratory directors may choose to temporarily refer some work to other laboratories until improvements to their own facility have been made.

Laboratory Inspection
Engineering controls Administrative controls Personal protective equipment General Laboratory Safety Laboratory self-inspection is the systematic examination of all safety features and processes within the laboratory (engineering controls, personal protective equipment and administrative controls). It helps to ensure that: Proper engineering controls are being used and are functioning adequately as designed Personal protective equipment is appropriate for the tasks being performed Appropriate site and protocol specific administrative controls are in place Decontamination of waste and materials has been adequately considered and proper waste management procedures are in place Proper procedures for general laboratory safety, including physical, electrical and chemical safety are in place. Biosafety practices and procedures are also examined. Laboratory self-inspection is an on-going quality and safety assurance activity that should take place on a regular basis. Adequately trained safety and health professionals may conduct laboratory self-inspection activities. Clinical laboratory facilities may develop audit, survey or inspection tools to help ensure consistency in the self-inspection process. Findings of the self- inspection should be discussed with laboratory personnel and management. Within the laboratory, an individual should be identified and made responsible for ensuring that corrective actions are taken for all deficiencies identified.

Risk Assessment Will evaluate all procedures for risks related to aerosol generation and injury from contaminated sharp objects (e.g., needle sticks) and develop a strategy for safe, step-by-step manipulation of both specimens and cultures. Laboratory activities required for the evaluation of a patient with possible tuberculosis include: specimen collection; transport of specimens to the laboratory; verifying labels and logging in specimens; initial processing that may include transferring specimens to tubes for centrifugation; preparation, staining and reading of smears; preparation of specimens for culture; and preparation of isolates for further study, including antimicrobial susceptibility testing. Risk Assessment- An action or series of actions taken to recognize or identify hazards and to measure the risk or probability that something will happen because of that hazard. This is different from a self-inspection because it 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, and the likelihood that such exposure will cause a laboratory acquired infection. Through the process of risk assessment, the laboratory environment and the work to be conducted are evaluated to identify hazards and develop interventions to ameliorate risks.

Why an assessment. Who should perform it. When should it be done
Why an assessment? Who should perform it? When should it be done? What tools are used? How should it be done? All workers are entitled to a safe and healthy working environment. The laboratory is responsible to develop its own guidelines and work practices to ensure a safe work environment for all employees. To develop effective strategies to continually guarantee employees a safe work environment, performance of risk assessment must be an integral and on-going part of lab operations. Why? To provide information needed to keep people safe- people in the immediate lab, the entire facility, community. Effective use of resources Identification of training needs and supervision Advanced planning for renovations Evaluation of procedural changes Prevent biohazard transmission to family of employees Justification for space and equipment needs Cost effective laboratory operation Evaluation of emergency plans Who? Shared responsibility- managers and employees- Best are those closest to the situation When? At regular intervals, at least annually, and with changes- move, renovation; new equipment, new reagent, new infectious agent What Tools? Lab record review- injury and surveillance reports, equipment maintenance, employee training, environmental monitoring Inspection- daily monitoring by employees, periodic walk through Review published materials- equipment manuals, manufacturer bulletins/newsletters, product inserts, scientific journals, public safety manuals and guidelines Observed lab operation- new procedures, new employees, new equipment, workflow Other- background, experience, common sense, ability to visualize potential outcomes How? First, identify the hazard, second determine the degree of risk. Begin with the arrival of the specimen at the facility not limited to the immediate laboratory. Physical facility- air-flow, lab access, furniture, floors, comp of water Containment equipment- BSC, centrifuge/cups/carriers, transportation carriers Personnel- experience and training, physical handicaps, attitude, immune status Types of procedures- aerosol generating, use of sharps, temperature extremes, dexterity and sterile techniques

Potential Hazards: The following slides show some improper biosafety practices or containment safeguards found in some TB laboratories. I found it scary that in the largest of the laboratories that we visited, ones that processed hundreds of specimens per day, had technologists working without respirators, some also worked without gloves. I’ve seen techs washing gloves with disinfectant while sitting at the hood to keep from changing them. I witnessed a tech reach into a pocket with a gloved hand, pull out an old tissue, blow her nose and put it back in the pocket and continued working with patient specimens for TB testing. Sinks without soap and/or towels. Some sinks located on the other side of an adjacent room. I am now going to show a collection of photos taken from some of our visits. Some of these have been corrected since our reports were released. When walking through the laboratory try and observe without making the techs self conscience. These should be used for training purposes not for penalty. Share results with laboratory administration in addition to the staff. Poor workflow- collision of workers (spills, dropping) Opening containers outside of BSC (contamination or cuts) Poor BSC practices (disrupt airflow will escape for BSC) Failure to check equipment before use Bunsen burner use- airflow disruption, infectious aerosols from flaming wet slides, burns Overfilled sharps containers Spread of hazardous materials from gloves Improper disposal of waste Unfamiliar procedure, equipment, reagents I have a collection of photos from laboratory visits. I will not identify the laboratories and am using them for instruction. I do not mean to demean the laboratories shown in any way. Many of the problems noted were corrected after our visits and reports were issued.

Lab is in a separate room but has open windows and an improper centrifuge.

Good placement of supplies but no room between BSCs and second BSC is used to store equipment.

Equipment to be decontaminated is left on an open bench to be performed later.

Standing rinse water.

No respirator, gloves, short sleeves

Large volume, no gloves respirators, gowns

No gloves, sleeves rolled up.

Open air staining should be set up where air does not flow across slides to tech.

No masks, or gowns. Chair height not adjusted correctly, feet not supported.

Unsecured tank too close to door.

Supplies stored in hood.

Overcrowded BSC.

Unsafe container position

Centrifuge pushed to the back would be difficult to see into to look for breakage.

Tubes correctly loaded in BSC.

Filter on the left is new
Filter on the left is new. The filter on the right was used in the door to the TB lab for 1 week. Makes you wonder if you should be protecting the lab from the outside air instead.

Summary Although the incidence of tuberculosis is higher in laboratory workers than for the general population, the risk of becoming infected with M. tuberculosis in the laboratory can be minimized through the use of the engineering controls, administrative procedures, and specific work-place practices that are presented in these guidelines. Full biosafety level 3 is recommended for laboratories performing work with live tubercle bacilli that may generate infectious aerosols. Currently available procedures for preparing AFB smears, preparing samples for culture, identification and antimicrobial susceptibility testing of AFB all have the potential for generation of aerosols and must be done using BSL-3 practices and procedures. Biosafety level 2 facilities and procedures are sufficient for laboratories performing direct AFB smears on samples that have been treated to inactivate the tubercle bacilli.

Questions?

Resources www.psmile.org www.asm.org www.clsi.org www.cdc.gov
cations/biosafety/en/Biosafety7.pdf

Biosafety in the TB Laboratory

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