LABORATORY AND FIELD SAFETY EQUIPMENT FOR THE MANIPULATION OF HIGHLY INFECTIOUS AGENTS
Center for Disease Control, Public Health Service, U.S. Department of Health, Education, and Welfare, Atlanta, Georgia 30333, U.S.A.
Hazard group 4 viruses, such as Ebola, Marburg, and Lassa, cause serious disease in man, with high mortality, for which there is neither specific cure nor prevention. These viruses, although basically natural zoonotic agents, can be transmitted directly from person to person. Those persons at highest risk appear to be workers engaged in the diagnosis of the disease and the care of patients. Such work often must be done in circumstances where elaborate systems for patient isolation, personnel protection, and disposal of infectious wastes are not available. Evaluation of the specific biohazards involved is a prerequisite for any discussion of specific measures designed to prevent infection among persons exposed to these viruses.
CONTAGIOUSNESS OF EBOLA, MARBURG, AND LASSA VIRUSES
Although little is yet known concerning the contagiousness of Ebola, Marburg, and Lassa viruses, two simple facts have been elucidated and serve as a framework for practical action. The first is that large amounts of virus are present in the blood and in many of the tissues of persons infected with any one of these three viruses. The second is that person-to-person transmission of these viruses by means other than parenteral contact is not common. As documented elsewhere in this symposium, person-to-person transmission was only 5-13% for Ebola virus in Zaire and Sudan. Although precise data are not available for Marburg and Lassa viruses, the fact that these agents have not become epidemic or pandemic in the absence of zoonotic transmission means that they are not, in fact, highly contagious. Were any of them to spread by droplets as do the influenza viruses, for example, our world would surely and literally be a very difficult one in which to live today. This is not to say that we can ignore the droplet route of transmission. Circumstantial evidences of such secondary infection are on record, but almost no experimental work has been done on this general problem.
In summary, I believe that the major hazard in dealing with patients suspected of having hazard group 4 infection is that of sub-integumentary exposure
to blood, secretions, and excretions. Infectious aerosols are a less likely risk. Approaches to these problems will be discussed in terms of primary and secondary containment.
Basic personnel protection can be achieved through scrupulous use of disposable gowns, caps, shoe covers, and gloves. The object is to prevent liquids or semisolids from contaminating the skin, which may be broken. In addition to these garments, either full-face respirators equipped with High Efficiency Particulate Air (HEPA) filters or a combination of goggles and nose-mouth respirators similarly filtered should be used. Although such equipment is not comfortable to wear, particularly in hot, humid environments, and although it is unequivocally frightening to patients, their families, and people in their communities, it serves three critical purposes: it
1) protects the face and mucous membranes from direct contamination,
2) protects the respiratory tract against infectious droplets, and
3) prevents inadvertent autocontamination by touching the face, eyes, and other parts of the body with the hands.
Several details in the use of this basic equipment deserve mention. Gloves should be rinsed in disinfectant while being worn whenever they are grossly contaminated; they should also be rinsed in disinfectant before they are removed. The order in which clothing is removed is also important. The proper sequence is (1) shoe covers, (2) cap, (3) respirator-goggles (which should be stored in containers, such as plastic bags, and reused by a single individual), (4) gown, and (5) gloves.
The equipment and procedures outlined above should be standard for personnel working with patients and for those processing potentially infectious specimens in a field laboratory. Laboratory manipulations create further hazards; for example, a tube breaking in a centrifuge or aerosols generated during pipetting or trituration. Two additional, forms of protection are feasible under field conditions. 'The first is use of materials other than glass wherever possible. The second is use of a collapsible, flexible-film plastic isolator to provide a negative pressure, HEPA-exhaust-filtered environment for such work. Isolators which will contain small centrifuges and provide work space for two people are available in the United States for less than 1,500 dollars. Surface decontamination of the isolator and of materials removed from it is achieved by mopping and spraying 2% sodium hypochlorite or 5% peracetic acid solutions. These isolators were used successfully in 1963-64 in San Joaquin, Bolivia, during an epidemic of 700 cases of Machupo virus infection and again in 1976, at Kinshasa and Yambuku during the Ebola virus outbreak. Although electricity is required, the amount needed for such an isolator adds little to that required for other equipment, such as microscopes, lights, and a refrigerator or freezer.
An unavoidable hazard is that posed by the hypodermic needle. Its use should be minimized. Special needles with an antigravity flow valve and evacuated rubber-stoppered tubes should be used for obtaining blood specimens. Needles should be placed in disinfectant solution before being disposed of. Whenever syringes are needed, they should have a mechanism for locking the needle. For specimen collection, disposable equipment is always preferable to reusables.
Patients should be isolated from direct and indirect contact with other patients, visitors, and others working in close proximity. A separate facility, however humble, is the best solution. Access to this separate facility must be restricted to the minimum number of properly protected persons needed to provide clinical care. The same principles apply to a field laboratory. Where airflow from such an isolation facility cannot be completely controlled (the most common situation), distance and a favorable location in terms of prevailing winds from the nearest human dwellings are factors to consider and use to optimum advantage.
DECONTAMINATION OF REUSABLES
Primary soaking of instruments in 2-5% sodium hypochlorite is recommended. Beware of commercially available liquid concentrates, which may contain much less of the degradable active ingredients than shown on the label. Look for dry hypochlorite, which is sold for use in swimming pools. If kept dry, its strength will be as advertised. After instruments have been decontaminated in hypochlorite solution, they should be boiled for 20 minutes or autoclaved at 10 pounds pressure for 15 minutes (home pressure cookers will work) before they are washed, wrapped, and sterilized for reuse. Decontaminate with hypochlorite, then boil utensils patients use. Boil all cloth items which must be reused. Do these things in a special location away from the facilities used for other patients. Oil drums make excellent containers for such work.
DISPOSAL OF DISPOSABLES
The first rule for disposing of disposables is to contain such materials. Plastic bags, usually doubled, are the most convenient means of achieving this. Definitive disposal thereafter is a matter of burn and/or bury. Oil drums also make good emergency incinerators. Site them carefully and pay attention to the wind. Items to be buried should be buried deeply. This rule applies especially to cadavres. If plastic bags of sufficient size are not available, cloth strips soaked in phenol or formaldehyde can be used to wrap the body, and similarly soaked sheets can serve as an outer shroud.
TRANSPORT OF SPECIMENS
Principles for safe shipment of highly infectious specimens and specific methods for adhering to them are set forth in Packaging Note 695 of the Industrial Air Transport Association Tariff on Restricted Articles. Briefly stated, these principles call for a tightly sealed primary container, a sealed secondary container which has enough material, such as vermiculite or cotton, inside to absorb the specimen contents should the primary container leak or break, and a shipping container capable of resisting shocks generated by a free fall from 30 feet.
Whenever possible, primary containers should be of temperature-resistant plastic. Suggestions for labelling, documenting, and notifying addresses of packages containing such specimens are also described in the June 3, 1977, issue of the World Health Organization's Weekly Epidemiology Record.
Assuming that future disease outbreaks are caused by agents not significantly more contagious than Ebola, Marburg, or Lassa viruses, the materials and techniques outlined here should suffice to prevent infection in medical personnel and their contacts. I personally see no valid reason for quarantining such persons in the absence of an overt accident. There is as yet no evidence to suggest that persons incubating these diseases are highly infectious. In any event, it is important that one member of any team charged with investigation and management of an outbreak be responsible for all aspects of biosafety. That responsibility must be accompanied by unequivocal authority for obtaining compliance with the procedures to be followed.
Another question is whether individuals continually exposed to the viruses under consideration should receive passive antibodies to the virus. This, of course, presumes that the etiology of the outbreak has been established.
Detailed discussion of this subject is outside the scope of this paper. A cardinal rule, however, is that the use of such antibodies is not a substitute for adherence to appropriate safety procedures.
MAXIMUM CONTAINMENT LABORATORIES (MCL)
This subject will not be addressed in detail. It is evident, however, that the technology of modern virology exposes laboratory personnel to quantities of hazard group 4 agents which -are rarely or never encountered in nature. No manipulation of such viruses is risk free. Thus maximum primary and secondary environmental protection is mandatory. To protect the outside environment, all air must be specifically filtered and/or incinerated before it is exhausted. Similarly, liquid and solid wastes must be effectively decontaminated by heat or chemical means. Personnel must wear special clothes and take showers before leaving the laboratory.
Primary containment usually consists of a connected line of stainless steel, gas-tight cabinets, operated at an atmospheric pressure significantly negative to that in the laboratory. Double-doored autoclaves and chemical dunk tanks are employed to pass materials into and out of the cabinet line. Detailed requirements for such laboratories in the United States, United Kingdom, and Canada are set out in the Federal Register, Volume 41, Nº 131 of July 7, 1977, a handbook entitled Control of Laboratory Use in the United Kingdom of Pathogens Very Dangerous to Humans, issued by the Department of Health and Social Security, and the Medical Research Council Guidelines for the Handling of Recombinant DNA Molecules and Animal Viruses and Cells, respectively.
By now, it should be clear that the costs of constructing and maintaining such laboratories are quite large. They are even greater when one realizes that the worker unit output in the sealed cabinets is both quantitatively and qualitatively less than in ordinary microbiological laboratories. At present, MCL are functioning at the Center for Disease Control (CDC), Atlanta, the U.S. Army Medical Research Institute for Infectious Diseases, Frederick, Md., and the Microbiological Research Establishment, Porten, Down, England.
In an effort to address these problems, CDC has recently completed a new maximum containment laboratory in which the steel cabinet line is replaced by a plastic one-piece suit supplied with air from a source remote from the laboratory. Suits are chemically decontaminated in a special shower at the end of each work period. The cost saving is significant, and easy access is afforded to a much greater variety of equipment. A film depicting this laboratory is now available as a 16-mm sound movie or a 3/4-inch videotape from the national Medical Audiovisual Center, Atlanta, Georgia 30333, U.S.A.
Both primary and secondary protection against interhuman transmission of highly hazardous agents such as Ebola, Marburg, and Lassa viruses can be achieved under the most difficult field conditions. The major risk is inadvertent exposure to infectious blood, secretions, and excretions. Disposable clothing, respirators, sodium hypochlorite, heat, and disposal by deep burial, together with physical isolation of patients and field laboratories, are the principal tools required. The critical materials should be stockpiled in several centers for immediate availability.
A single individual should be given responsibility and authority for managing the safety program. The guidelines make it possible to replace irrational fear with a positive approach to containing such disease outbreaks without a public health catastrophe. Central to this thesis, however, is the concept that individuals can be found who believe that outbreaks can be contained. Biosafety in the hands of the morbidly anxious nonvolunteer is an invitation to disaster.
Long-term work with these viruses requires maximum containment laboratories (MCL) based on safety concepts so expensive that only three such facilities are currently operational. The use of self-contained plastic suits promises to reduce costs and permit a wide range of technology in the MCL of the future.
G.A. Eddy : In general I agree with the concept of a suit Laboratory and we have this on a limited scale at Fort Detrick. It certainly is a lot easier to work in a suit than it is in the class 3 system. We have also experience with Laboratory accidents but all of them occurred in association with the class 3 system. Regarding the handling of fluid waste. I think perhaps one way of handling that is to forget about autoclaving it. At least the viruses that we are working with don't have to be autoclaved in order to inactivate them. A reasonable alternative is the use of a high temperature short-time milk sterilizer. That technology has been very well worked up, they have all sorts of careful controls which provide recirculation in the event the temperature is not sufficiently high. That kind of apparatus is available at fairly reasonable cost. Dr. Johnson, what do you think about allowing a single person to work by himself in a suited system ? Would you permit one person to work by himself without anyone else in the Laboratory ?
K.M. Johnson : The answer is no. Of course the complication is double overtime for personnel in the weekends, if animals have to be fed. it is almost impossible for a single person to enter into such a suit, it is almost a two-men operation and our manual does call for at least two people at all times in the suit operation. We will continue to use our present cabinet laboratory far primary diagnosis on unknown materials, The reason is that, while we think that we can find chemicals that will clean our suits from known agents., that might not be true for everything in the world. Therefore our new laboratory will be primarily dedicated to investigation and reagent production for known agents all of which can be tested for chemical inactivation.
I.W. Pinkerton : By what means and how frequently would you attempt to decontaminate the laboratory room and would this put it out of action during any period of time ?
K.M. Johnson : It is planned to decontaminate the Laboratory rooms, both the suit Laboratory and the other one, with vaporized formalin. Eventually it is going to rattle all the equipment, think of ultracentrifuges. In some cases we may make a small compromise by protecting some of the equipment with plastic bags. Provided the animal section can be sealed off, the suit laboratory can be used the next day. The reason is that it has its own air supply
and residual formalin is no problem. For the cabinet Laboratory, it will take about two or three days for all the fumes to completely clear out.
T. Muyembe : What kind of equipment do you recommend for Laboratories in Africa? As you know, we don't have much resources and therefore we are looking for cheaper but adequate long-lasting equipment.
K.M. Johnson I certainly recognize, and we all must, that the infrastructural talents, money and systems that have to be available and have to be continuously maintained to keep one of these laboratories going are not available everywhere and they are not available, not only in Africa but in certain other places either. At the moment, I don't really think that I have an answer that I am willing to propose as such. On the other hand, if you are careful and you have carefully defined what the Limits of the work are going to be, it may well turn out that flexible plastic equipment such as you have seen or could see in the laboratory of Dr. Pattyn and Dr. Van der Groen here in Antwerp, may turn out to be acceptable and useful. What they have is a prototype, I should emphasize, and there has not been any significant amount of work done with class 4 agents in this type of equipment, except in the collapsable plastic isolators we used in the field years ago in Bolivia and more recently in Zaire for doing very simple manoeuvres. in neither of these situations, however, were we attempting to cultivate and grow viruses up to high concentrations. Whether the plastic approach will be feasible and up to what limit and what kinds of procedures for viral work, I don't think I can say. However in theory, I do not see any reason why not. Remember the history of the germ-free animal field, it was originally all stainless steel and heavy glass and eventually was converted into flexible films and it works. I think this could work as well. There are a number of specific problems that need to be addressed and tested thoroughly before one could say yes, I recommend it. I sympathize, I think it is something that deserves real attention because there is a major need, in the countries where these diseases are endemic, to be able to approach them.
R.E. Shope : Dr. Johnson, would you be willing to comment on your experience with the use of immune individuals in field laboratories in an area where you don't have a class 4 facility ?
K.M. Johnson : It's a beautiful solution to the primary containment problem, but it doesn't free you from having to be concerned about secondary containment. For example, this is exactly how we did all our work for ten years in the Panama Canal Zone, on a top floor laboratory which we adapted for secondary control at a very low price. Only people who were immune to Machupo virus were allowed to enter the Lab. All of them had acquired their immunity naturally in the course of field duty. We never had any break-throughs nor did we ever document booster responses in the staff. The main difficulty was of course that there were very few people in the world with such antibody. But you cannot assemble a group of people of limited education, train them and direct them from outside to do this work.
C.E. Gordon Smith : I think we are all looking forward with great interest to the assessment of your suit laboratory because it will overcome a number of problems we have all faced in the past. One important factor working in a cabinet is arm and hand fatigue that increases the risk factor. To reduce that would be very significant. I wonder if you could at this stage give an idea about the relative costs, capital and running of a suit laboratory as opposed to a cabinet laboratory.
K.M. Johnson : In many ways, the largest cost is in the system of secondary containment. Therefore the first decision is whether there will be autoclaving, boiling, or pasteurizing, or if it is going to be a dry laboratory. I would suggest that for the primary containment system, the initial capital cost is ten times less expensive than a cabinet laboratory. For maintenance, I think expenses are also lower. A further advantage is the basic flexibility in a suit laboratory so that it can be used for other purposes at a lower containment level if programme and future dictates. This is impossible for the cabinet laboratory.
R.A. Coutinho : Do you think that it is advisable that there would be a lot of laboratories that work on these viruses ? When something goes wrong with the laboratory, who is responsible ? Is it the company, the laboratory worker, the university, who is it ?
K.M. Johnson : That is going to depend very much indeed on all of those individuals and all those organizations that you listed as candidates to be responsible and I think it is going to vary from country to country. I don't think that whatever is done the person who scientifically is in charge of running the laboratory is ever going to escape being the primary victim. The commercial companies attempting to be of help in this field will probably do an honest job in trying to test the integrity of filters and all of their constructions, but it will be the user and purchaser of such equipment to have it constantly under his control. All this is the biggest reason why we still know so little about some of these viral diseases.
C.E. Gordon Smith : In setting out safety codes and instructions it is essential that it is clearly defined who is responsible for what, and that adequate responsibility is laid on the lowest members of the team because if they are not too responsible then the load for those higher up in the system is intolerable.