Safety in a Clinical Virology Laboratory
Safety in pathology is part and parcel of laboratory quality control. ISO has issued guidelines on safety in medical testing laboratories: ISO 15190. Although this guideline is not compulsory for the purpose of laboratory accreditation, all laboratories are recommended to follow these guidelines.
In this section, various aspects of safety in a clinical virology laboratory will be looked at. The topics covered are as follows:
Laboratory-Acquired Viral Infections
The following laboratory-acquired viral infections had been reported;-
Infections had occurred in widely different kinds of laboratories. Some of the organisms are handled only in research establishments whilst others are encountered daily in diagnostic and clinical laboratories. The vast majority of reported infections occur in research institutes although a wider population is at risk in a routine diagnostic laboratory. Laboratory-acquired infections are far more likely to occur in untrained workers. In laboratory-acquired infections, the route may not be the same as the natural route. Routes of infection reported are;-
The mains sources of laboratory-acquired infections are accidents, animals, clinical specimens, aerosols and work with the agent. The types of accidents involved consist mainly of spillage and splashes, needle and syringe, sharp objects and broken glass, animal scratch or bites.
Classification of Viral Pathogens into Hazard Groups
Micro-organisms have been classified into 4 hazard groups by the ACDP (Advisory Committee on Dangerous Pathogens) on the basis of pathogenicity to humans, risk to laboratory workers, transmissibility to the community, and whether effective prophylaxis is available.
Group 1. - An organism that is most unlikely to cause human disease
Group 2. - An organism that may cause human disease and which may be a hazard to laboratory workers but is unlikely to spread to the community. Laboratory exposure rarely produces infection and effective prophylaxis or treatment is usually available. Examples include herpesvirses, ortho and paramyxoviruses, picornaviruses, adenoviruses, unconventional slow viruses.
Group 3 - An organism that may cause severe human disease and presents a serious hazard to laboratory workers. It may present a risk of spread to the community but there is usually effective prophylaxis or treatment available. Examples include HIV, HBV, Hantaviruses, Japanese B encephalitis, Rift Valley fever, Yellow Fever, rabies.
Group 4 - An organism that causes severe human disease and is a serious hazard to laboratory workers. It may present a high risk of spread to the community and there is usually no effective prophylaxis or treatment. Examples include Lassa fever, filoviruses, smallpox, Crimean-Congo haemorrhagic fever, Russian spring-summer encephalitis, Kyasanur forest.
Where pathogens that cause severe human disease are known to infect by the airborne route, primary containment using microbiological safety cabinets and the provision of secondary containment using appropriate ventilation have been recommended. For work with bloodbourne organisms such as HIV and HBV, the use of "sharps" should be avoided. Where available, vaccination is recommended for people working with known organisms. Vaccination is essential for work with HBV, polio, rabies, yellow fever, Rift valley fever, Russian spring-summer encephalitis, Kyasanur Forest disease virus. Vaccination is recommended for measles, mumps and rubella.
Containment requirements for work on hazard group 4 specimens
Hazard group 4 pathogens include viruses that cause encephalitis and viral haemorrhagic fevers. The Health and Safety (Dangerous Pathogens) Regulations 1981 stipulate that the Health and Safety Executive must be notified 30 days in advance before a diagnostic service in relation to a listed pathogen is carried out.
Viral Haemorrhagic Fever
Clinicians should consider the possibility of a VHF in a person developing a febrile illness within 21 days of arrival from an endemic area or a laboratory worker in a containment level 4 laboratory or animal unit presenting with an unexplained fever. However, the main life-threatening but treatable microbial diseases are malaria and typhoid fever. Therefore, allowance should be made for the diagnosis of malaria and the performance of certain biochemical and haematological investigations without the need for more rigorous physical containment than necessary. The guidelines developed by the ACDP are graded depending on the level of suspicion.
Minimal Risk - these are patients who have come from cities where the risk of VHF is negligible. No laboratory work must be carried out on specimens from these patients until a blood film has been examined for the presence of malaria parasites. The blood film should be rendered safe at the bedside by fixing in formalin or methanol. If blood is sent to the laboratory, a Class I microbiological safety cabinet must be used. If malaria parasites are not demonstrated, the categorization of the patient should be reassessed, and if infection by a Hazard Group 4 pathogen is still a possibility, specimens must be handled in a Containment level 3 laboratory using a Class III microbiological safety cabinet.
Moderate Risk - these are patients where infection with a Hazard Group 4 pathogen is a distinct possibility on epidemiological grounds but the more likely clinical diagnosis s malaria. No laboratory work must be carried out on specimens from these patients until a blood film has been examined for the presence of malaria parasites. The blood film should be rendered safe at the bedside, if blood is sent to the laboratory, it must be a Containment level 3 laboratory and a Class I microbiological safety cabinet must be used. If malaria parasites are not demonstrated, the categorization of the patient should be reassessed, and if infection by a Hazard Group 4 pathogen is still a possibility, specimens must be handled in a Containment level 3 laboratory using a Class III microbiological safety cabinet.
High Risk - these are patients where infection with a Hazard Group 4 pathogen is suspected e.g. the patient has been living and working in an endemic rural area, as opposed to an urban area, in particular a medical worker, or works in a laboratory where Hazard Group 4 pathogens are in use. If the patient has worked in a laboratory where Hazard Group 4 pathogens are in use, the patient should be immediately transferred to a high security unit. No laboratory work must be carried out on specimens from these patients until a blood film has been examined for the presence of malaria parasites. The blood film should be rendered safe at the bedside, if blood is sent to the laboratory, it must be a Containment level 3 laboratory and a Class III microbiological safety cabinet must be used. If malarial parasites are demonstrated and VHF as a diagnosis is discarded after assessment by clinical and laboratory staff, specimens may then be handled in routine clinical laboratories. If malarial parasites are not demonstrated, or if malarial parasites are demonstrated, but infection by a Hazard Group 4 pathogen is still a possibility, specimens for non-virological work which is essential before the patient is transferred to a specially designated high security isolation unit must be handled in a Containment Level 3 laboratory in a Class III microbiological safety cabinet.
Laboratory Procedures for suspected Hazard Group 4 Pathogens - Specimens for Hazard Group 4 virus serology or isolation must be sent, by arrangement to the Virus Reference Laboratory, CPHL, where work will be carried out at Containment Level 4. Specimens for the diagnosis of other viral infections may be sent to other laboratories that comply with the requirements for Containment Level 4, but only with their consent. Material for transport to a Containment Level 4 laboratory must be packed according to instruction given by the receiving laboratory. When there is a likelihood of many hours delay before a patient is transferred to a unit designated for the management of VHF cases, specimens for non-virological tests which are essential for the management of the patient may be carried out in Containment Level 3 laboratory in a Class III microbiological safety cabinet. Potentially infectious material to be removed from a Class III safety cabinet for incubation or storage must first be placed in hermetically sealable containers which are disinfected with 2% glutaraldehyde or Hypochlorite before removal. Such hermetically sealed containers must not be reopened unless they have been transferred back into a Class III microbiological safety cabinet. Sealed units must be used for centrifugation, and they must be opened only in a Class III safety cabinet. All waste materials and discarded clothing must be rendered safe to handle before they are removed from the laboratory and autoclaved before disposal or recycling. Water used for hand washing must be rendered safe by either chemical or hear disinfection. A high security isolation unit for the management of VHF would have appropriate Containment Level 4 laboratory facilities on-site.
Viral Encephalitis due to Hazard Group 4 Pathogens
Some Hazard Group 4 Pathogens which cause encephalitis (e.g. Russian spring-summer encephalitis) may arise in a patient or laboratory worker. Specimens for virus diagnosis should be handled in a Containment Level 4 laboratory as above. Specimens for other clinical investigations should be handled in a Containment Level 3 laboratory in a Class III microbiological safety cabinet.
There is no evidence that blood from patients with clinical rabies contain rabies virus. Virus may, however, be present in saliva, tears, urine, CSF and tracheal aspirates for at least two weeks after the onset of symptoms. Therefore, haematological and biochemical investigations on blood specimens may be carried out in a laboratory that complies with the requirements of work at Containment Level 2. When non-virological investigations on any specimens other than blood are required, the work must be undertaken in a Containment Level 3 laboratory and a Class I safety cabinet should be used. Staff performing this work must be vaccinated against rabies. Gloves should be worn for all manipulations which should be undertaken with care, and where possible, without the use of needles or glassware, to avoid the possibility of skin puncture. If automated equipment is used, it should be disinfected after use by washing through with an effective disinfectant compatible with the system. Specimens for virological and antibody testing for rabies should be sent to the Virus Reference Laboratory, CPHL.
Creutzfeldt-Jacob Disease is a transmissible spongiform encephalopathy (TME). In recent years, there has been an increase in iatrogenic cases through injections of human cadaver derived growth hormone, cornea grafts, dura mater grafts for eardrums, and contaminated neurosurgical instruments. TME agents are highly resistant to heat, radiation and chemical inactivation. The sterilization and disinfection procedures for TME agents are far more stringent than those usually applied in hospital practice for the control of conventional bacterial, viral, and other infectious disease agents. Contaminated reusable instruments should be sterilized at 134-138oC for not less than 18 minutes (hold time) or for six cycles of 3 minutes. It is thought that the downward displacement autoclave which is commonly used in laboratory work may be less effective even at these high temperatures than the surgical sterilizer operating with a pulsed vacuum cycle. Scrapie-infected tissue stored in 10% formalin has been shown to be still infectious. The application of sodium hypochlorite containing 20000 ppm appears to be effective when used in decontaminating surfaces. 2M sodium hydroxide may be used instead for the decontamination of metal surfaces that may be corroded by hypochlorite. Phenolic disinfectants, B -propiolactone, glutaldehyde, formaldehyde, alcohols and ethylene oxide are regarded as unreliable. There is practical difficulty in disinfecting safety cabinets of TME agents because of their resistance to formaldehyde and other fumigants.
To date, there are no confirmed cases of laboratory-acquired infection. However, the long incubation period makes the cause and effect difficult to relate and two recently reported cases of CJD in ex-laboratory workers who had handled neural tissues extensively are a matter of concern. Therefore, particular caution is advised in dealing with tissue from the CNS, especially from suspected cases of CJD and GSS. Equipment used in the laboratory and post-mortem room should be subjected to stringent disinfection procedures. Work practices should be reviewed to avoid puncture wounds and cuts. Eye protection and gloves should be worn. The skull of a CJD or GSS patients should only opened in a bag. At present, level 2 containment is advised for TME agents with additional measures to guard against puncture wounds and cuts and contamination of broken skin and eyes. The following precautions are recommended by the ACDP and WHO;-
Decontamination should be carried out by hypochlorite 20,000 ppm for at least 1 hour, repeated wetting with the disinfectant is necessary over the treatment period. 2M Sodium hydroxide is also active against scrapie but may not completely inactivate high concentrations of agents, as in the case of hypochlorite, repeated wetting is necessary. Autoclaving should be carried out at 134oC for 18 minutes as a single cycle, or as six separate cycles of 3 minutes each at 134oC.
Classification of laboratories according to use
It is generally accepted that three or four kinds of microbiology laboratories are required. In the WHO classification these are; Biosafety Level 1 to 4 for Risk Groups 1 to 4 microorganisms respectively.
1 2 3 4
Isolation of laboratory No No Op Yes
Room sealable for decontamination No No Yes Yes
inward air flow No Op Yes Yes
mechanical via building system No Op Op No
mechanical, independent No No Op Yes
filtered air exhaust No No Op Yes
Double door entry No No Yes Yes
Airlock No No No Yes
Airlock with shower No No No Yes
Effluent treatment No No No Yes
Autoclave on site Yes Yes Yes Yes
in laboratory room No No Yes Yes
double ended No No Op Yes
Biological safety cabinets
Classes I or II No Yes Yes Op
Class III No No Op Yes
A problem which should be mentioned is that although these classifications may be reasonable for clinical and research laboratories, they are hardly applicable to many industrial, food science and agricultural laboratories.
Animal House Containment and biosafety levels
The rules for animal houses parallel those for laboratories. Consideration should be given to the requirements of individual species such as their behavior, zoonoses and parasites.
|Level||1||As for Level 1 laboratories plus limited access, arthropod control, protective clothing and gloves|
|Level||2||As for Level 1 laboratories and animal houses plus decontamination of waste and cages before washing, microbiological safety cabinets and personal protection devices|
|Level||3||As for Level 2 laboratories and animal houses plus controlled access|
|Level||4||As for Level 3 laboratories and animal houses plus strictly limited access, waste decontaminated before removal, and clothes changing room and shower|
The Safe Working Environment
Basic Levels 1 and 2 laboratories
Although Containment Levels 1 and 2 laboratories are considered to be adequate with microorganisms which offer minimal risk to the worker. The potential for such risks is enough to merit design features additional to school laboratories and offices. A safe working environment implies enough working space. In the UK, it is recommended by the DHSS that each worker should have at least 18.6 m2 of space. It is now generally accepted that each worker be given 3 m run of benching and 24 m of free air space (ACDP). Access to laboratory areas by people who do not work in them should be strictly limited. Members of the general public should get no further than the reception areas or waiting rooms.
In the interest of safety, floors should be slip resistant, seamless, be impermeable to liquids and resistant to most, if not all chemicals that are normally used in laboratories. The surfaces of walls and partitions should be smooth, impervious and easily cleaned. Windows should be sealable and fitted with blinds. Doors should be fire resistant and fitted with vision panels. Ceilings also need to be impermeable and should be coved to the walls. Openings in walls and ceilings for the entry of pipes etc. should be sealed around such pipes. Bench surfaces should be impervious to liquids, and not easily corroded or stained by chemicals. Electricity and gas supplies to the benches are needed, water and waste plumbing is optional. Each laboratory should have a hand basin and disposable paper towels provided.
Ventilation systems designed to prevent the distribution of infectious airborne particles were developed during the Second World War based on the "Clean-to-Dirty" airflow principle, where more air is extracted from the rooms where hazardous materials are handled than from any other area. Close-fitting doors are used and thereby resulting in a pressure gradient so that air always flows from clean to potentially contaminated areas ie. from corridors to laboratories and not in the opposite direction. The air extracted from contaminated areas may be ducted directly to the atmosphere. It is important that adequate lighting is provided. It is not usually necessary to fit microbiological safety cabinets into Levels 1 and 2 laboratories.
Level 3 laboratories
The object of level 3 laboratories is to confine, or contain the organisms so that only a minimum number of people are exposed to them. Hence the policy of designating a whole microbiology department as a level3 laboratory is fatuous. A true level 3 laboratory is suitable for one or two persons only. If the amount of work merits more staff, than separate Level 3 laboratories should be provided. All the design features advocated for Level 2 laboratories apply. Although Level 3 laboratories may open off non-public corridors, it is best if access is from other laboratories of a lower Containment Level. Access to Level 3 laboratories should be strictly limited and controlled and the doors should be locked when the rooms are not in use. Microbiological safety cabinets are essential features of these laboratories. Care is needed in siting those in relation to airflows and staff movements. An incubator room could open directly from a Level 3 laboratory and there should be enough storage space e.g. refrigerators and deep-freezers so that Hazard Group 3 organisms need not be kept elsewhere.
Level 4 laboratories
Work with Hazard Group 4 agents is usually severely restricted in most countries by government decree. Therefore a great deal of consultation and supervision is necessary in the planning and building of these laboratories. The laboratory should be isolated or physically separated from other parts of the same building so that access is difficult. It should be airtight and access is through airlocks. The ventilation system should be completely controlled so that air flows via air locks into the laboratory. Class III safety cabinets should be maintained at a lower pressure than the room. All effluent air is passed through double banks of HEPA filters before discharge to the atmosphere. A double-ended autoclave is essential to ensure that nothing passes outside the room without being sterilized.
Specimen reception rooms
A specimen reception room should be separated from offices and treated as a potentially infected area. The floors and surfaces must likewise to leak-proof and amenable to decontamination procedures. A hand basin is essential and access should be restricted to authorized persons.
Preparation or utility rooms
One or more of these rooms should be designated for the reception, treatment and disposal of contaminated waste. The design features should be those of a Level 2 laboratory equipped with autoclaves, a sluice, a waste disposal unit plumbed to a public sewer, deep sinks, glassware washing machines, drying ovens, sterilizing ovens and large benches. These should be arranged to preclude any mixing of contaminated and decontaminated materials.
Walk-in incubators and refrigerators
The microbiological hazards associated with these concern firstly, their distance from the laboratories, and secondly raised thresholds. Spillages and breakages of cultures in corridors are much more hazardous than in rooms. Raised thresholds are sometimes fitted to incubator and refrigerator rooms to accommodate floor insulation. There is little need for floor insulation since the average concrete floor will not lose or gain enough heat to affect an incubator or cold room.
Microbiological Safety Cabinets
Microbiological safety cabinets are designed to capture and retain infected airborne particles released in the course of work and to protect the laboratory worker from inhaling them. Until recently, safety cabinets had an unrivaled record of poor installation and misuse. There are three classes of safety cabinets.
Class I - air is drawn from the room through the open front, and over the working area. It is then passed through high efficiency particulate air (HEPA) filters which remove infectious particles, and is ducted to outside air. A minimum airflow of 0.7 m/s must be maintained through the front of the cabinet. Filters must be changed when the airflow falls below this level.
Class II - air is filtered and most of it is recirculated through the cabinet. This cabinet protects the work as well as the worker. About 70% of the air is recirculated through filters so that the working area is bathed in clean (almost sterile) air. The remaining 30% of air is exhausted to the atmosphere and is replaced by a "curtain" of room air which enters at the working face.
Class III - Class III cabinets are totally enclosed and leak-proof. The operator works with gloves which are sealed into the front of the cabinet by removable gaskets.
Laminar flow (clean air) cabinets - these are not microbiological safety cabinets. Air is drawn through HEPA filters and passed onto the working surface and the room. They are widely used in pharmacies and the preparation of tissue culture.
The siting of safety cabinets are important. The main problems are caused b draughts from doors and windows and the movement of people and therefore, safety cabinets should not be sired near doors. HEPA filters are highly efficient in removing viable microorganisms with a quoted efficiency rate is 99.997%. It is important to ensure that ducted effluents are not discharged near to open windows, especially of hospital wards
Class I Microbiological Safety Cabinet
Class II Microbiological Safety Cabinet
Class III Microbiological Safety Cabinet
Decontamination of safety cabinets
The working surfaces and walls of safety cabinets should be decontaminated on a day-to-day basis by swabbing with disinfectant. Glutaraldehyde is probably the best disinfectant for this purpose as phenolics may leave sticky residues and hypochlorites may, in time, corrode the metal. For thorough decontamination e.g. after spillages, before maintenance, filter changing and testing, fumigation is necessary. This may be done by formalin or vaporized glutaraldehyde. Before fumigation, the installation should be checked to ensure that none of the gas can escape to the room or other rooms. Formaldehyde is generated by boiling formalin or by heating paraformaldehyde.
The Laboratory Worker
Protective Clothing - Over the years, three kinds of protective clothing came into general use: gowns, front-buttoned white coats and specially designed overalls which afforded protection up to the neck. The latter is probably the best protective clothing available since they are easy to remove, aesthetically pleasing, and afford much better protection than front-buttoned white coats. It is advisable to wear disposable plastic aprons over ordinary protective clothing when working with blood that might be infected. Gloves should be worn when handling all Group 3 agents.
Amenities - Hand basins should be provided in each laboratory, lockers for staff clothing should be available near to but not in laboratory rooms, and rest rooms for eating and drinking be provided near to laboratory rooms.
Health of staff - it is advisable to have some sort of pre-employment medical examination. The form of the examination may include a medical history, physical examination, FBC, clinical chemistry, urinalysis, ECG and chest X-ray. It is desirable to have some sort of medical monitoring for workers in Containment 3 and 4 laboratories. The scope of the monitoring depends on the agent. Women who work with viruses should tell their supervisors as soon as they are pregnant. There should be an individual evaluation of the risks involved in continued employment and it may be necessary for them to change their work. Limited information is available about laboratory exposure to CMV, the ACDP does not mention exclusion from any kind of work but does offer general guidance.
Accidents - There should be a rule in all microbiological laboratories that all accidents causing personal injury involving infectious agents or not should be reported to the supervisor or safety officer. In the UK, there is a statutory requirement for the reporting of all serious accidents to the Health and Safety Executive. Records should be kept of all staff sicknesses, injuries and accidents, and of X-rays and immunization. Accident records are particularly important for legal reasons. It is highly desirable to have a qualified first-aider in the laboratory, but he should receive additional training in dealing with exposure to and ingestion of infectious, toxic, and corrosive chemicals. All staff should know who to contact if an accident occurs. The standard first aid kit for minor injuries should be in a prominent position and well sign-posted. Care should be taken with eye irrigation since the bottles of "sterile" water are frequently contaminated.
Vaccination - in choosing which vaccine to give to the laboratory worker, several considerations should be taken into account, such as (1) the frequency with which the organisms concerned are encountered in the laboratory, (2) the known incidence of infection with those organisms, (3) the severity of the disease they may cause, and (4) who handles them. A large number of vaccines are now available for viruses, which include rubella, mumps, measles, and polio, rabies, Yellow Fever, VEE, TBE, Rift Valley, and Russian spring summer encephalitis.
Minimizing Equipment and Technique-Related Hazards
Mouth pipetting must be banned at all times. Pipetting devices should be inspected routinely for leakage and services and replaced as necessary. When discharging infectious material from a pipette, it is important to minimize the formation of aerosols; The last drop in the pipette should not be forced out but left in the pipette, care should be taken to avoid bubbling when mixing liquids with pipettes, it may advisable to discharge the liquid onto the side of the container. Contaminated pipettes should always be discarded into disinfectant fluid which is prepared daily. Broken and chipped pipettes should be discarded.
2. Hypoderemic needles and syringes
These are the most hazardous pieces of equipment in common use. Surveys have shown that at least one-quarter of overt accidents which result in infection were caused by these instruments. Many inoculations result during experiments with animals, others occur during transfer of infected material by syringes. Many needlestick accidents occur when the needle is being disconnected from the syringe so that blood may be discharged into its specimen container gently to avoid haemolysis. Some hospitlas and institutions ban disconnection, but other solutions are available such as needleguards which permit the safe disconnection and recapping of needles, needle forceps, and vacuum collection outfits. It is important that used needles are disposed properly into harden "sharps containers" which are then autoclaved before disposal. These "sharps containers" should be available where they are needed.
Infectious material may be dispersed by a centrifuge either through broken tubes or other means such as through the threads of the tubes and caps. Sealed centrifuge buckets should be used when centrifuging infectious material. Care should be taken to ensure that the centrifuge tubes are not cracked or flawed. Tubes should not be more than three-quarters full, especially if angle centrifuges are used. Tubes should be capped and they and the buckets should be balanced carefully to avoid vibration which may lead to breakage. Material containing agents which are particularly likely to cause laboratory infections should centrifuged in sealed centrifuge buckets which are subsequently opened in a safety cabinet.
4. Pouring infectious material
Pouring of the supernatant after centrifuging cultures or viruses, NPA and diarrhoeal specimens is common practice. These fluids are usually poured into disinfectant. Two hazards may arise: production of aerosols, and contamination of the outside of the tube from which the material is poured. These hazards may be minimized by pouring infected fluids into a funnel, and that small strips of blotting paper be used to wipe the rims of tubes after pouring.
Collection, Transport and Receipt of Infectious Materials
A large number of specimens are transported by various means between different hospitals and laboratories everyday. During transit, the containers or the packages enclosing them may be handled by many people. The containers may be involved in accidents and be damaged or broken. They may also be lost or stolen and opened by unauthorized persons. Although there are no reports of infections acquired this way, it follows that any infectious material being transported could present a hazard to any person in contact with it. Therefore, the collection, packaging, transport and unpacking should be strictly regulated. The postal and transport authorities of different countries have different regulations governing the transport of infectious materials.
Collection of Specimens
Containers - In most laboratories, disposable, screw-capped 25 ml bottles have replaced glass containers. Specimen containers must be sufficiently robust to withstand the stresses they are likely to meet and leakproof. Reusable containers (e.g. glass) must remain leakproof during their lifetime. They should be inspected before reassembly.
Blood - traditionally blood is collected with hypodermic syringes and needles and then expelled into plastic containers. There are problems with the stoppers of a lot of these containers which result in external contamination. Needle stick injuries are particularly likely to occur in resheathing the needle and this practice should either be discouraged or a safer method of resheathing be used, perhaps with the aid of certain newly developed devices. Vacuum collection tubes minimize several of the hazards and problems of taking blood and disposing safely of syringes and needles. These containers are robust, leakproof, and cannot be contaminated externally.
Faeces - faeces are probably the most hazardous material other than blood. The specimens are often collected by the patient who is rarely instructed on the proper method of collection. Patients tend to fill the container with faeces so that fermentation may lead up to a build-up of gas during transport. This may result in leakage or the cap coming off, moreover, when such as container is opened, a violent broadcast be result. The other possibility is that the outside of the container ma be contaminated. Patients should be told to defaecate on a pile of toilet paper in the lavatory pan and to remove a pea-sized portion with the spoon provided, to insert it carefully into the container and then to screw the cap on tightly.
Other Specimens - sputum specimens are probably less hazardous to handle because of the decreasing incidence of tuberculosis in developed countries. Apart from some viruses, the organisms present in sputum are rarely infectious. Nevertheless, the contamination of the outside of the container remains a problem. The risks from urine are minimal, although a small proportion of patients may excrete salmonella typhi, leptospires, hepatitis or HF viruses. It is debatable, therefore, whether any expensive precautions are needed in the case of urine. External contamination is a problem and there is a strong case for collecting urine from females in sterile large jars before transferring to a laboratory container. Other specimens such as pus and aspirated fluids are collected by professional staff which minimizes the risk of external contamination.
Labelling specimens and containers
Labels and stickers used should be self-adhesive for obvious reasons. There is much controversy about "Danger of Infection" and "High Risk" labels. These are often used for specimens where there is a special risk such as HBV or HIV. The problem is that these labels may give a false sense of security to the staff who should any specimens as potentially infectious with dangerous pathogens. Specimens labelled "Danger of Infection" should be placed in self-sealing plastic bags. Pins and staples should not be used as they may prick the hands of any persons handling the bag. Request forms should be separate from the specimen.
Transport of Specimens
Transport within hospitals - the DHSS code of practice stipulates that leak-proof (bottoms and seams should not leak) trays or boxes should be provided for the transport of specimens from wards to the laboratory, and these should be able to withstand autoclaving or overnight exposure to disinfectant. Staff carrying the specimens should wear overalls and encouraged to hand wash frequently. The taking of specimens into canteens and kitchens should be forbidden.
Transport between hospitals - the HSAC (1991) requires that "special" secure transport boxes with secure lids. These must be capable of withstanding autoclaving and prolonged exposure to disinfectants. These boxes should be inspected daily for leakages and decontaminated and washed out at least weekly.
By inland post - the Post Office Guide (1986) stipulates that Hazard Group 4 agents must not be posted. Hazard Group 3 material must have a "Danger of Infection" label on the container (but not on the outside of the box). The container used must be robust and cocooned in enough absorbent packing material to retain the contents in the event of leakage or damage to the container. It is wise to place the container in a self-sealing plastic bag although this is not required by the Post Office. The material must be sent by first class mail.
By air mail or air freight - before any material is sent to another country by air mail, the sender and receiver must ascertain that it is acceptable to the authorities of that country. The material should be placed in a watertight "primary container" which is then packed in a watertight "secondary container" and the whole packed in an "outer container" All containers must be approved by the postal authorities. The following documentation are required; (1) An International Air Traffic Associations Shippers Certificate for Restricted Articles, (2) Labels giving contents, description and quantity of infectious substance, (3) Green customs label, (4) Official address label.
Receipt of infectious material
There is clearly a difference between the hazards posed by packages sent to a specialist or reference laboratory and those to a routine diagnostic laboratory. The former are likely to contain cultures or concentrates of infectious agents whereas the bulk of the latter is not particularly infectious. It is advisable that cultures and such specialized materials are unpacked in the laboratory by professional staff. There is concern over the use of clerical staff for receiving and documenting specimens. It is not unusual to see food and drink being consumed by clerical staff near the specimens. The disturbing large number of untrained staff who acquire infection in the laboratory undoubtedly include clerical and reception staff. Therefore it is essential that clerical staff handling specimens should be given some form of training in the safe handling of specimens. Any specimen in a plastic bag which carries a Danger of Infection label should not be removed from that bag. The accession number can be put on the outside of that bag. Leaking or broken specimens should not be touched, nor should any others in the same box or tray. Provision should be made for a member of the professional staff to deal with them. These specimens should not be allowed to stray to other parts of the room.
Contaminated laboratory waste
Laboratory waste should be separated into colour-coded containers. The recommended colours are;-
Yellow - for incineration
Light blue or transparent with blue inscription - for autoclaving (but may be incinerated subsequently
Black - normal household waste: local authority refuse collection
White or clear plastic- soiled linen
There are three practical methods of treating contaminated laboratory waste: sterilization by autoclaving, chemical disinfection, and incineration. The first two are laboratory processes; incineration involves the transport of material off-site. Sterilization and disinfection are not synonymous: sterilization implies the killing of all microorganisms; disinfection kills most microorganisms but depend greatly on the chemical used.
Autoclaving involves the timed exposure of materials to steam above atmospheric pressure and hence at temperatures above 100oC. Autoclaves operate at high pressures and temperatures and their manufacture, installation, and use are regulated. In the UK, they are subjected to the Pressure Systems and Transportable Gas Containers Regulations 1989, which requires regular inspection and maintenance and regular checks on the effectiveness of the sterilization cycles. For most purposes, sterilization is achieved by exposure of material to 121oC for 15-20 min. Higher temperatures such as 126oC for 10 minutes, or 134oC for 3 minutes are permitted. Temperatures are monitored by thermocouples in modern autoclaves. Chemical and biological indicators are also widely used, the most well-known of which is the "autoclave" tape. Biological indicators is common use consist of strips or discs of filter paper which have been soaked in suspensions of Bacillus stearothermophilus spores, then dried and packed in porous envelopes or sachets. These are then placed in various positions in the load and after processing, the strips and discs are removed from their packing and placed in nutrient broth which is incubated for 24-48 hours. The presence of turbidity indicates failure to sterilize.
It was customary to place material for autoclaving In wire baskets and cylindrical buckets. While these containers are perfectly acceptable for bottled fluids, they are not suitable for potentially infected material since they do not contain spilled material. Therefore, shallow buckets or other "solid-bottomed containers" should be used for sterilizing infected material which may leak. It is important to ensure that the container used is not overlarge since it may lead to inadequate sterilization. Autoclavable plastic bags are now in general use and they must be permeable to steam. The mouths of plastic bags should not be closed before autoclaving since this will reduce steam penetration.
Chemical disinfection should be regarded as a first line defence especially in the case of discarded bench equipment, which should be followed by autoclaving or incineration. There is a rough spectrum of susceptibility of microorganisms to disinfectants. The most susceptible are vegetative bacteria, fungi, and enveloped viruses. Mycobacteria and non-enveloped viruses are less susceptible and spores are generally resistant. The most commonly used disinfectants in laboratory work are clear phenolics and hypochlorites. Aldehydes have a more limited application and alcohol mixtures are less popular. Iodophors and quaternary ammonium compounds (QAC) are more popular in the US than in the UK.
1. Clear Phenolics - these compounds are effective against vegetative bacteria (including mycobacteria), fungi, enveloped viruses (including Lassa and Marburg). They are inactive against spores and non-enveloped viruses. Most phenolics are active in the presence of considerable amounts of protein but are inactivated to some extent by rubber, wood, and plastics. They are not compatible with cationic detergents. Laboratory uses include discard jars and disinfection of surfaces. Clear phenolics should be used at the highest recommended concentration. Dilutions should be prepared daily and diluted phenolics should not be stored for more than 24 hours. Skin and eyes should be protected.
2. Hypochlorites - the active ingredient is chlorine, which is very effective against vegetative bacteria (including mycobacteria), spores, fungi, and both enveloped and non-enveloped viruses. Hypochlorites are considerably inactivated by protein and to some extent by natural non-protein material and plastics. They are not compatible with cationic detergents. Laboratory uses include discard jars and surface disinfection but they corrode some metals and thus care is necessary. They should not be used on the metal parts of centrifuges. For general purposes and discard jars, 2500 ppm is recommended. For spillages of blood and discard jars which may receive much protein, 10000 ppm should be used. The hypochlorites sold for laboratory applications in the UK contain 100000 ppm. Hypochlorites decay rapidly in use and diluted solutions should be replaced after 24 hours. Hypochlorites may cause irritation of skin, eyes, and lungs,
3. Aldehydes - formaldehyde (gas) and glutaraldehyde (liquid) are good disinfectants. They are active against vegetative bacteria (including mycobacteria), spores, fungi, and both enveloped and non-enveloped viruses. They are active in the presence of protein and are not very much inactivated by natural or man-materials or detergents. Formaldehyde is supplied either as a solid polymer, paraformaldehyde, or as a liquid, formalin. Both forms are heated to liberate the gas which is used for disinfecting enclosed spaces such as safety cabinets and rooms. Glutaraldehyde usually requires an activator, such as sodium bicarbonate. Most activators contain a dye so that the user can be sure that the disinfectant has been activated. Effectiveness and stability after activation varies with product and the manufacturers literature should be consulted. Glutaraldehydes are particularly useful in disinfecting metal surfaces as they do not cause corrosion. They may also be used in discard jars but they are comparatively expensive. Aldehydes are toxic and precautions must be taken when formaldehyde is used.
4. Alcohol and alcohol mixtures - alcohol and alcohol mixtures are effective, albeit slowly, against vegetative bacteria and enveloped viruses. They are not effective against spores, fungi and non-enveloped viruses. They are not inactivated by protein and other material or detergents. Alcohols and alcohol mixtures are useful for disinfecting surfaces. They are relatively harmless to skin but may cause eye irritation.
5. Quaternary ammonium compounds - they are cationic detergents that are effective against vegetative bacteria and enveloped viruses and some fungi. They are not active against mycobacteria, spores, and non-enveloped viruses. They are inactivated by protein and a variety of natural and plastic materials and soap. Their laboratory uses are therefore limited but they have the distinct advantages of being stable and non-corrosive to metals. They are usually used for cleaning surfaces and are also very popular in food hygiene laboratories because of their detergent nature. They are non-toxic and harmless to the skin and eyes.
6. Iodophors - like chorine compounds, these iodines are effective against vegetative bacteria (including mycobacteria), spores, fungi, and both enveloped and no-enveloped viruses. They are rapidly inactivated by protein, some natural and plastic substances and are not compatible with anionic detergents. Laboratory uses include discard jars and disinfecting surfaces. They can also be used for handwashing since they usually come with a detergent. Iodophors are relatively harmless to the skin by some eye irritation may occur.
Precautions in the use of disinfectants - As indicated above, some disinfectants have undesirable effects on the skin, eyes, and respiratory tract. Disposable gloves and safety spectacles, goggles, or a visor should be worn by anyone handling strong disinfectants. Full-face respirators should be worn when rooms are being fumigated with formaldehyde.
Discard Jars - discard jars should be robust and autoclavable. Glass jars should not be used because they are easily broken. Polypropylene beakers or jars are probably the most serviceable items. Wide-moth screw-capped polypropylene bottles or jars are better still. They are left open on the bench but are closed at the end of the working session. It is important that discard jars are filled frequently with known active dilutions of disinfectant, not overloaded with protein or floating articles. Laboratory supervisors should ensure that inappropriate articles are not placed in discard jars e.g. a 25ml pipette has no place in a small 1 litre jar. Funnels may be used to prevent splashing and aerosol dispersal. Discard jars should be washed with hot water before re-use. It is better still to autoclave the jar before washing. The contents of the discard jars should then be autoclaved and/or incinerated.
Re-usable pipettes - after use, re-usable pipettes should be completely immersed in disinfectant so that no air remains. They should remain in disinfectant for at least 18 hours before washing and/or autoclaving.
Disinfection after accidents
There is an immediate need for disinfection after spillage, breakage or other accidents involving infectious material. The safety officer should draw up protocols for decontamination procedures after particular accidents. Action to be taken ranges from simple mopping up with disinfectant to decontamination of large items of equipment or of whole rooms. Appropriate equipment should be available for all such emergencies.
1. leakage of specimens - this is of particular concern when it happens in reception rooms with clerical staff. Saving the specimen, which may not be repeatable may be considered as important as protecting the staff. Leakages should be dealt with by a member of the laboratory staff. Disposable gloves should be worn and the offending container and any others which may be contaminated be placed on a tray which is then placed in a large plastic bag and removed to a laboratory, preferably a safety cabinet. The surface on which the leakage occurred should then be covered with paper towels over which the appropriate disinfectant is poured. This should be left for at least 30 minutes before the towels are removed. The area should then be swabbed with fresh disinfectant and left for a further 30 minutes before it is dried. The tray used should be flooded with disinfectant and left for several hours. Leakage into transport boxes should be treated in the same way except that the box can be autoclaved.
2. Simple spillages - simple spills should be covered promptly with a paper towel and disinfectant poured gently on the towel. The towel should removed after 30 minutes and the are swabbed with fresh disinfectant. If material is spilled in a safety cabinet, the fan should be left running while the spill is being dealt with. After a spillage, the safety cabinet should be disinfected by formaldehyde.
3. Serious spillages - the most serious spillages are those that involve cultures dropping on the floor and break. There is much splashing and a considerable amount of aerosol is dispersed. Laboratory workers should not bend down to inspect the damage. Instead, all people in the room should hold their breath and leave. Any possibly contaminated clothing should be removed. If that is not practical, then the affected area should be sponged with a disinfectant-detergent. Hypochlorites should not be used because it will bleach the fabric. The door should be closed and a warning sign posted onto it. Thirty minutes is a reasonable time to allow the aerosol to settle or clear. The safety officer should by this time have ascertained the possible nature of the organism involved and decide what respiratory or other protection is required when re-entering the room, which disinfectant to use, and the extent of the room decontamination procedures e.g. from mopping up to total decontamination of the room by fumigation
4. centrifuge accidents - breakage of tubes in a centrifuge can disperse large amounts of aerosols. Sealed buckets should be used for all Hazard Group 3 agents. The action to be taken is much the same as that for the breakage of a culture. After the room is considered safe to enter, the centrifuge buckets and rotor can be removed and autoclaved and the bowl disinfected.
5. Transport associated accidents - these may range from leakage of a pathological specimen in a hospital van to leakage or breakage of a culture in a public place, post office, public service vehicle or aircraft. In principle, all accidents can be treated as above for decontaminating surfaces and rooms. However, since the potential for the spread of infection is so great, it is essential to inform the senior medical microbiologist and environmental health officer at once. Protocols for dealing with these emergencies have been worked out by most health departments and have been published by the WHO.
1. Rooms - it may be necessary to disinfect a room after a spillage of infectious material, before a change of use or before building alterations or redecoration. Two methods may be used; wet disinfection and fumigation. Wet disinfection is only applicable if the floors do not allow liquids to leak into the rooms below and any remaining furniture or equipment is not likely to be damaged by disinfectant. The floor is mopped by a disinfectant and then left wet for at least 30 minutes. It may also be possible to disinfect the whole room with a sprayer. The operator should wear eye and face protection and if formalin is used, breathing apparatus should be worn. Fumigation of whole rooms is now not generally recommended because of health threats. If fumigation is carried out, then it is of utmost importance to ensure that there is no possible leakage from the room. Windows, ventilators, pipe runs and cracks should be sealed with masking tape. The operator must have full eye and face protection as well as a respirator. He should leave the room immediately after starting the reaction. The door should then be locked and sealed with masking tape and a warning notice be posted and the room left for 24 hours.
2. Equipment - it is desirable to have a regular procedure for decontaminating equipment that is to be serviced on site or returned to a manufacturer for any reason. Many companies require certificates that state specifically that the equipment is microbiologically safe. The interiors of incubators, refrigerators and similar cupboard-like equipment should be swabbed thoroughly with glutaraldehyde or alcohol-formalin mixture and left overnight. It should be washed out several times with clean water and dried. After decontamination, the equipment should be taped or otherwise secured so that it cannot be used and labelled. In the case of automated equipment, the effluent discharge tube should be taken at least 25 cm into he laboratory waste plumbing system or that it is trapped in a bottle. The instrument is then flushed through with a disinfectant recommended by the manufacturer.
3. Laboratory protective clothing - laboratory protective clothing should be autoclaved before it is sent for laundering or placed in "hot wash" bags which should not be opened in laundries until they have been exposed to very hot water.
Disposal of contaminated liquid waste
Small amounts of contaminated liquid waste may be poured into disinfectant but problems arise with large amounts such as urines, effluents from automated equipment, and sink and lavatory effluents from Maximum Containment laboratories. It is safe to pour most specimens down a sluice or deep sink which will join the public sewer. Sewage disposal plants deal quite effectively with most pathogens. The only hazard to the operator is from splashing and aerosol production when pouring and this should be done with care. Afterwards or at the end of the day, the sluice or sink should be flushed with disinfectant. If it is known or suspected that the urine contain Hazard Group3 pathogens, neat disinfectant (hypochlorite or phenolic) should be added to the urine. The effluents from automatic apparatus are unlikely to be very hazardous since microorganisms are likely to have encountered inimical chemicals on their way through the machine. The safe disposal of the various effluents from Containment Level 4 laboratories is a design consideration. The effluents are usually taken to one or one holding tanks where they are heated by steam for a predetermined time, then tested for the presence of pathogens before discharge into the sewers. Sometimes, chemical treatment is preferred.
Precautions Against Hepatitis and HIV Agents
A "universal" approach should be adopted for the prevention of infections by blood and body fluid-borne organisms such as HBV and HIV. To-date, there have only been two cases of laboratory-acquired HIV infection; one attributable to needlestick injury and the other to exposure to a virus culture supernatant fluid. Numerous cases of laboratory-acquired HBV infection had been recorded through contact with infected blood or body fluids. The primary mode of infection is contact between the hands and the infected material where the virus enters through cuts and abrasions. There is no evidence that the inhalation of aerosols of blood or other body fluids plays any part in laboratory-acquired HBV infections but this could certainly not be excluded as a possibility.
Where standard precautions are used, there should be a set of standard operating procedures and only skilled staff should be employed. They should have received HBV vaccine. The WHO, but not some other authorities recommends medical surveillance and base-line serum samples.
1. Precautions against contact - protective clothing should be worn at all times and may be supplemented by plastic aprons. Good quality disposable gloves should be worn. Cuts, scratches and abrasions on the hands should be covered. Masks, visors, or goggles should be worn if there is a possibility that the face may be splashed with blood or other material. Methods for the collection and transport of specimens should be reviewed to reduce as far as possible the number of operations that expose the operator and the possibility of leaking containers. Blood may be splashed when containers are opened, and suitable precautions should be taken e.g. gripping the stopper or cap through a strip of paper wrapped around it confines dispersal. Blood specimens should be centrifuged in sealed centrifuge buckets. If serum is separated by pouring, the rim of the tube should be wiped with filter paper soaked in hypochlorite or glutaraldehyde. Care should be taken in making and handling blood films. Sampling probes of automated apparatus should be wiped with tissues held in gloved hands.
2. Precautions against injection - accidental inoculation with hypodermic needles, other sharp instruments and broken glass may be reduced by replacing such hazardous equipment with inherently safer articles. Needles may be replaced by cannulas, glass pasteur pipettes by the plastic disposable type, and other glassware by rigid plastic or break-resistant glass. All re-usable glassware should be examined before it leaves the preparation room for chips and rough edges. Used sharp objects and broken glass should be discarded into rigid containers. Special precautions should be taken against needlestick accidents. Care is also essential when approaching the sharp sampling probes of some automated instruments.
3. Action in case of accidental exposure - Instructions for action should be part of the SOPs. If the skin is accidentally punctured or that skin lesions are contaminated, immediate action is necessary. Bleeding should be encouraged under running water, a dressing applied and the accident reported and appropriate follow-up action taken. The affected part should be washed immediately with soap and water. Mucous membranes and conjunctivae should be thoroughly irrigated with water.
4. Automated equipment - automated equipment should be of the "closed system" type that is capable of being decontaminated by passing disinfectant through it. The probes should be shielded to avoid splashing. Effluent should be collected in closed bottles or discharged at least 25 cm into the waste plumbing system.
5. Inactivation of specimen - some attention has been given to the possibility of inactivating the viruses in blood and other specimens so that they are safer to work with. The most commonly used method is heating the specimen to 56oC for 30 minutes which is sufficient to inactivate HIV to undetectable levels . However, this is unlikely to have any effect on hepatitis B, which is thought to require temperatures of up to 80-90oC for inactivation. Other methods such as treatment with ethanol or with B -propiolactone has been advocated but doubts had been cast on the effectiveness of all three methods. Buffered Forman may be useful for decontaminating blood films. In the case of histopathology specimens, small specimens such as needle biopsies will be fixed and decontaminated quickly, whereas larger pieces of tissue may require several days. If frozen section work is unavoidable, the cryostat should be well shielded and the operator should wear face and eye protection. After use, the machine should be brought to room temperature and disinfected.
6. Spillage, decontamination and disposal of waste - if blood or other infected material is spilled, the spillage should be immediately mopped up with gloved hands, and disinfected using either glutaraldehyde or hypochlorite (10000 ppm). HIV is effectively inactivated by alcohol mixtures, hydrogen peroxide, hypochlorites and paraformaldehyde at the strengths usually employed in laboratories. Contaminated protective clothing should be autoclaved or bagged for hot wash laundering. Gloves should be discarded into contaminated waste receptacles for autoclaving or incineration. If street clothing is contaminated, it should be sponged with hot detergent solution, possibly with glutaraldehyde depending on the nature of the material.
Infection Hazards of Human Cadavers
Cadavers may pose infection hazards to people who handle them, such as pathologists, nurses, mortuary attendants, embalmers, funeral directors and members of the emergency services. Viruses which poses particular risks include HIV, HBV, HCV, CJD, rabies, yellow fever and viral haemorrhagic fevers. The following practices are routinely carried out on cadavers;-
1. Bagging - the body is placed in a plastic bag. This is recommended for cadavers infected with the above high risk viral agents. A problem with bagging is that it speeds up decomposition since it slows down the cooling of the cadaver.
2. Viewing - the bereaved is allowed to see, touch and spend time with the body before disposal. This is not advised for high risk agents.
3. Embalming - chemical preservatives are injected into the body to slow the process of decay. Cosmetic work may be included. This is not advised for high risk agents although embalmers were thought to be at low risk due to the wearing of protective clothing and the use of formalin-based preservatives.
4. Hygienic preparation - the body is cleaned and tidied before viewing. Some ethnic groups requires this process to be carried out by close relatives. Again this is not advised for high risk agent.
In general, following COSHH (Control of Substances Hazardous to Health) precautions, especially the use of protective clothing will greatly reduce the risk of acquiring infection. Some additional precautions may be advisable for particular infections.
Hepatitis B and C;- HBV is extremely infectious and health care workers especially those in morbid anatomy are particularly at risk. Embalmers are also thought to be at risk from needle stick injuries. Therefore the bodies of those known to be infected with HBV should be handled only by workers wearing full protective clothing. Mortuary workers and embalmers should be vaccinated against HBV. HCV is transmitted by the same routes as HCV but probably less infectious. Therefore, the same precautions should be taken.
HIV;- HBV and HIV are transmitted by similar routes and thus the precautions for HBV such as full protective clothing should be adequate to prevent the transmission of HIV. To date, no embalmers or mortuary technicians had developed infection following documented exposure although a few may have acquired the infection occupationally. HIV survives for many days after death in tissues preserved under laboratory conditions.
CJD;- The agent that causes CJD has been shown to survive well in formalinised tissue. Exposure to sodium hypochlorite containing 20,000 ppm for 1 hour, or autoclaving at 134oC for at least 18 minutes are needed for decontamination. The Health and Safety Commission suggested recently that skulls of people who have died of CJD should only be opened inside a large plastic bag fitted over the head and neck of the cadaver.
Reduction of Risk
The Howie report (1978) presented a detailed code of practice for the prevention of infection in laboratories and necropsy rooms. There are detailed protocols for the layout, construction, ventilation and operation of necropsy rooms, Hands should be washed routinely after each procedure and the environment cleaned with a phenolic disinfectant daily. The instruments should be washed in a washer-disinfector, autoclaved or immersed in a phenolic disinfectant for 20 minutes. A phenolic disinfectant is preferred to hypochlorite because hypochlorite is corrosive and may damage surfaces and instruments.
At present, funeral directors are not given access to the death certificate and therefore they may not be alerted to potential infection hazards. Body bags are becoming more and more commonly used but they may be unpleasant to the relatives. All instruments used for embalming or preparing the body for the funeral should be cleaned and warm water and detergent and then disinfected by a phenolic disinfectant. Although an autoclave provides excellent decontamination, this is not justified by the existing levels of risk.
Role of Safety Officers
All laboratories should have at least one person who is well-versed in safe practices and who understands and can advise the management on its legal responsibilities in respect of the health and safety of its employees. Under the UK Health and Safety Regulations 1988, employers have a legal obligation to appoint as safety advisors "competent" persons who will advise and assist in protective and preventive measures against accidents and ill health caused by their work. The suggested duties of a safety advisor (officer) include:-
One or more SOPs should be drawn up for dealing with:
In the drawing of an emergency SOP, the following should be taken into account;-