EVALUATION OF THE PLASMAPHERESIS PROGRAM IN ZAIRE
K.M. JOHNSON, P.A. WEBB, D.L. HEYMANN
Center for Disease Control, Public Health Service, U.S. Department of Health, Education and Welfare, Atlanta, Georgia 30333, U.S.A.
The therapeutic value of human convalescent plasma (passive-antibody) in the treatment of patients with highly lethal viral diseases remains generally unproven. Reports on its successful use are mainly anecdotal. One instance where it was thought to be of value in the treatment of a laboratory acquired arena virus infection has been published (1). A laboratory worker developed Lassa fever while conducting experiments with animals infected with the virus. Five hundred ml of plasma with a titer of 1:64 by complement fixation (CF) was administered on the 10th day of illness, after which marked clinical improvement was noted; in 24 hours temperature returned to normal and viremia dissappeared. Virus was recovered from throat washings 4 days after plasma was administered and from the urine on days 5 and 22 after plasma therapy. Antibody was not demonstrated until approximately 7 weeks after onset of illness. The authors concluded that the administration of plasma with high titer of antibody was beneficial in aborting the disease.
Eddy et al. (2), using an experimental monkey model, have described the use of immunoglobulin in the prophylaxis and treatment of another arenavirus infection, Bolivian hemorrhagic fever. They demonstrated protection against development of initial clinical illness following Machupo virus infection in rhesus and cynomologous monkeys by using Machupo immunoglobulin of human origin given either before or shortly after virus inoculation. The finding must be viewed with caution, however, because some survivors developed severe neurological signs one to two months after virus inoculation and died 4-6 days thereafter. The data suggested that neurological sequellae were associated with high doses of immunoglobulin rather than intermediate or low doses. Although the efficacy of therapy remains undefined, in the face of the devastating epidemic in Zaire of a new virus disease with such an alarmingly high fatality rate, one early priority was to identify survivors with high levels of antibody and to obtain plasma which might be used for treatment of future patients and team members who might become infected during the investigation. The success of this program hinged upon rapid identification of donors, and the Critical determinant was the ability to test for fluorescent antibody (FA) in the field. This capability permitted bypassing the logistic logjam of shipping specimens to the base laboratory, in this case, to the U.S.A., and the inevitable delay before results could be returned to the field.
The first units were obtained in Kinshasa from three convalescent patients who had been transported there for plasmapheresis. Ten days after the first unit was obtained in Zaire, the efficacy of its use was tested in a laboratory worker accidentally infected with Ebola virus. The outcome was clinically gratifying and this gave impetus to a major plan to obtain more plasma by transferring operations to Yambuku, the epicenter of the disease. Dr. Courtois has told you how that program was carried out.
Table 1 shows the number of donors and units per donor obtained. Note that over half of the 201 units collected were obtained from six individuals.
The first and last units obtained from each donor were titrated for FA in the Atlanta Laboratory, and only four units from three donors, had titers of less than 1:64 by the indirect FA technique.
Tests run simultaneously on serial bleedings from six people disclosed that peak titers ranging from 1:256 to 1:1024 occurred 1-2 months after onset of disease. One to two months later most donors had experienced some decrease from peak titers. Seven months after onset of illness, all of the 13 persons tested still had antibody titers of 1:64 or higher.
All donors were found to have microfiliarial parasitemia. Tests for hepatitis B surface antigen (HBs Ag) on the first and last units from each donor were negative.
SUMMARY OF COLLECTION PATTERN, EBOLA ANTIBODY-CONTAINING HUMAN PLASMA, KINSHASA AND YAMBUKU, NOVEMBER, 1-JANUARY 25, 1976-1977.
1 - 4
5 - 9
10 - 14
15 - 21
Units with IFA titer gte 1:64 - 197
Ebola immune plasma was given to one team member suspected of having acquired an Ebola infection. The cause of his illness has not been determined; however, Ebola infection was ruled out. The decay curve of passive antibody in the individual is shown in Table 2. After 500 ml of plasma with an antibody titer of 1:256 was administered, circulating antibody of 1:32 was noted for approximately two weeks, with decreasing titers and disappearance by three months.
DECAY CURVE OF PASSIVE ANTIBODY ADMINISTERED TO INDIVIDUAL WHO DID NOT HAVE EBOLA VIRUS INFECTION
Day after plasma
Indirect FA titer
DISPOSITION AND PROCESSING OF PLASMA
Currently, plasma is stored in the frozen state (-20º) in Yambuku, Kinshasa, Johannesburg, and Atlanta. No definite decision has been made regarding further processing. Tentative evidence suggests that the following problems may influence the final decision :
1. Antibody given in the native unaggregated state intravenously might provide maximum effect.
2. Known uptake and half-life of ordinary gamma globulin which is aggregated and cannot be given intravenously (i.v.) is shorter than for unaggregated antibody (3,4).
3. The yield of antibody in gamma globulin is definitely a function of the starting plasma volume.
4. Methods for preparing gamma globulin suitable for i.v. use are still experimental in the U.S.A. Such procedures involve at least 30% further loss in antibody content as well as shortening the half-life in vivo (3,4)
5. Long-term storage of antibody at refrigerator rather than freezer temperatures is highly desirable for stability.
One possible approach to all of these problems would be simple lyophilization of plasma either in pools from individual donors or in a master pool from a few selected donors. Pooling of plasma would provide a uniform product as far as antibody titer is concerned and would permit better evaluation in a treatment program, but pooling would increase the risk of contamination by hepatitis B virus, since the radioimmunoassay (RIA) technique is not infallible. Some of these problems will be addressed in a later section of the colloquium. A final tribute must be paid to those Zairois people who, in the face of this terrifying disease, still gave their blood in the hope that others might be saved.
The authors thank Dr. Kenneth R. Berquist, Phoenix Laboratories Division, Center for Disease Control, for performing the HBs Ag tests on plasma samples.
Two hundred and one units of convalescent plasma containing Ebola virus antibody were obtained from 26 donors. Most of the units had titers of 1:64 or higher. All units were HBs Ag negative, but all were found to contain microfilaria. The units are being stored at -20ºC in four different localities. Plans for future processing are discussed. The key factor in the success of this program was the ability to detect antibody-positive persons in the field by using partially inactivated antigen slides and a fluorescent microscope.
1. Liefer, E., Goche, D.J., Browne, H. (1970) Lassa fever, a new virus disease of man from West Africa. II Report of a laboratory-acquired infection treated with plasma from a person recently recovered from the disease. Amer. J. Trop. Med. & Hyg., 19, 677-679.
2. Eddy, G.A., Wagner, F.S., Scott, S.K., Mahlandt, B.J. (1975) Protection of monkeys against Machupo virus by the passive administration of Bolivian haemorrhagic fever immunoglobulin (human origin) Bull. WHO, 52, 723-727.
3. Waldmann, T.A., Strober, W., Blaese, R.M. (1970) Variations in the Metabolism of Immunoglobulins Measured by Turnover Rates, in Immunoglobul Biological Aspects and Clinical Uses, Natl. Acad. Sci., pp. 33-48.
4. Knoblet, H., Barandun, S., Diggelmann, H. (1967) Turnover of standardgammaglobulin, pH,4-gammaglobulin and pepsin desaggregated gammaglobulin and clinical implications. Vox Sang. 13, 93-102.
A. Fabiyi : In our efforts to collect plasma for Lasso, we have always measured the blood protein Level before we bled any individual. in order to relieve donors from further dangers, instead of going to an area where facilities are not available, efforts are made to transport them from one area to another, a few hours by Jeep, to a base where all the facilities for the plasmapheresis are available. We were just informed that the presence of microfilaria in the plasma is no contraindication for the use of it. What happened to the team member to whom plasma was given ? Were there any problems in that regard ?
K. Isaäcson : The patient has been given heated plasma, so I don't think the microfilaria posed any problem.
J. De Smyter : where is the convalescent plasma being kept now ? Will some be processed into gammaglobulin or do you consider this wasteful ?
K.M. Johnson : With respect to the plasma obtained in Zaire, Dr. Webb indidated the places without specifying exactly how many ml in each. It is presently distributed in the original plasma state. The question of further processing of the plasma in order to stabilize the antibody over a long period of time. and lots of other problems, is very complex and will be discussed later on.
J.G. Breman : Dr. Simpson, of the 46 positive donors at the first bleeding how many were included in the 59 donors that Dr. Draper had in his group and of those 59 how many persons were negative that were positive in the first group ?
D.I.H. Simpson : They were all, I think, included in the second round and I think only two had become negative.
J.G. Breman : However, there were 23 units in the second bleeding of the 101 taken that were negative.
D.I.H. Simpson : They did not all have two units taken off. When I say 23 units, some of them were from patients who were not patients at all, but who had been included in Dr. Draper's list because the hospital thought that these were cases, they were not the documented cases that we had.
T. Muyembe : How much plasma was obtained in Zaire, and how much of it was left in Zaire ? What are the indications for its administration ? We frequently have alarms, which are perhaps false alarms, but one is tempted in these circumstances to administer the plasma.
D. Courtois : in Yambuku 201 units of plasma were obtained. Dr. Johnson will be able to tell where they are now. The problem of the indication of its administration is extremely delicate, for it is very rare and should be used accordingly. it should be administered in the early days of the disease. Clinical diagnosis of the disease is difficult and can only be made in an epidemic context. We have been faced with this problem for one of the team members in Yambuku. It took us 3 days to make a decision. And we had no precise and rapid diagnostic criteria. In the future there will be many false alarms and frequently the decision will have to be bade at a distance. The only good criterium is the epidemic context. The decision on an isolated case will always be rather unscientific.
K.M. Johnson : in the case of the Zaire plasma, the agreement with the Minister of Health at the time was that half of it was destined to stay in Zaire and subfractions of the other half were to be kept both in Europe, somewhere, and in the United States, when a final decision is made on the type of processing, if any. The amount obtained represents at the moment a unique pool of antibody. We had that one chance, we seized it and it is very clear today that you couldn't go back and do it again no matter how much money anybody would put into it. So there is a very great concern that this antibody be kept in the state that will best preserve it for the longest possible time so that it can be used where it is needed.
P. Brès : in this connection, it should be noted that plasma cannot be administered to haemorrhagic fever patients before the etiologic virus has been antigenically identified. Remember that the plasma from Johannesburg given in Kinshasa did not do any good because the two viruses were antigenically different. This means that in the future morphological diagnosis of an Arena or an Ebola-like virus based on electron microscopy will not be sufficient but that the agent needs to be antigenically identified which may take another eight or fifteen days.
K.M. Johnson : I couldn't agree with you more, the question of a rapid highly specific viral diagnosis is not just an academic game.