RESULTS OF EBOLA ANTIBODY SURVEYS IN VARIOUS POPULATION GROUPS
G. VAN DER GROEN , K.M. JOHNSON , F.A. WEBB , H. WULFF , J. LANGE
1. Prince Leopold Institute of Tropical Medicine, Laboratory of Virology, Nationalestraat 155, B-2000 Antwerpen, Belgium.
2. Center for Disease Control, Atlanta, Georgia 30333, U.S.A.
Antibodies to Ebola virus were measured by the indirect fluorescent technique (IFA) (1). Virus infected monolayers of Vero cells were trypsinized when 20-40% of cells were infected and the suspension of cells was exposed to ultraviolet light (1200-1400 microwatts HS/CM2) for 15 minutes. Cells were dropped onto wells in teflon templated slides, fixed in acetone for 10 minutes then frozen at -60ºC until used. Our original purpose was to identify convalescent persons who might be suitable as plasma donors. Thus IFA tests were initially done in Zaire, and serum samples were sent on to Atlanta for confirmation. Eventually a total of 39 persons was judged to have Ebola antibodies. 33 of these were originally detected in Zaire, and only one serum scored as positive there failed to be confirmed in Atlanta. Thus we believe that use of the IFA method is practical in the field if electricity and a freezer, dry ice or liquid nitrogen are available. Use of microscope with a halogen light source and epicentric illumination simplified technical problem in setting up for work
The results of Ebola IFA on human sera done to date are shown in Table 1. Initial work done on persons who were ill or had close contact with fatal Ebola cases in the Yambuku region suggested that such antibody was uncommon but uniformly of at least 1:64 titre when present. Seventeen percent of persons who experienced an acute illness after such contact had such antibodies. Subsequent extension of the serosurvey, however, disclosed that a few persons with or without contact with a case of hemorrhagic fever and no symptoms also were positive. Additionally there were small numbers of individuals whose area had low titres of IFA to Ebola virus. Additional positive sera were found in villages where no fatal case had occurred. These individuals had not visited cases in other villages nor the Yambuku mission hospital. Four of the 5 persons with titres of 1:64 or greater were rebled in June, 1977 and three of them were still positive for Ebola antibodies. One further high titered and two low titered sera were collected by Dr. Peter Piot in early 1977 during a retrospective evaluation of a fatal case of possible hemorrhagic fever in Libela (38 km south of Yambuku).
In January 1977 a small epidemic occurred in Northern Bosogo (Uganda). Twenty three patients were affected, 10 persons died. Signs and symptoms included high fever, headache, sore throat, cough and pharyngitis, pneumonia. Three patients had a maculo papular rash over the abdomen, 2 cases developed diarrhoea, melena and haematemesis. One serum of a female taken 16 days post onset of fever showed Ebola IFA titre of 1:4, an inconclusive but probably negative result.
Serological results on human sera collected from convalescent patients, case contacts and controls during the Ebola outbreak in Nzara and Maridi townships of Southern Sudan in the second half of 1976, are indicated in Table 1 2 with the permission of E.T.W. Bowen and colleagues
Sera from two "control" groups also were tested. We found 6 of 243 sera obtained in 1975 from northern Rhodesia had IFA, one to a titre of 1:32. Samples from 200 Panamanian San Blas Indians were tested with results shown in Table 1.
Our conclusions from these data are as follows
1. The mortality rate of Ebola infection in Zaire was high; very few sur vivors were detected when specific antibodies were used as the criterion.
2. Asymptomatic infection was unusual but did occur.
3. An IFA titre of 1:64 was a good, but not a perfect index of past infection.
4. Ebola virus may be endemic in Zaire, as shown by persistent antibodies in a few persons with no apparent case contact during the epidemic.
5. Another method for measurement of typespecific antibodies is needed. To date virus plaques under agar have not been observed, and neutralization of virus-induced cytopathic effects has been hampered by the virus breakthrough phenomenon even when serum "accessory factor" was added.
1. Wulff, H., Lange, J.V. (1975) Indirect immunofluorescence for the diagnosis of Lassa fever infection, Bull. W.H.O., 52: 429-436.
2. Bowen, E.T.W. et al. (1978) Virological studies on a case of Ebola virus infection in man and in monkeys (this symposium).
EBOLA IFA ANTIBODIES IN HUMAN SERA FROM SELECTED GEOGRAPHIC REGIONS
Number of Samples tested
Total number of positive anti-Ebola IFA sera
IFA titre 1:64
IFA titre 1:32 - 1:8
IFA titre 1:4
* Villages (Yaeto, Likau, Bokoy, Nbengbe, Yandongi Moke) which had no fatal cases of hemorrhagic fever
** 29 members of Maridi school who had no history of recent illness and had not been in contact with known or suspected cases of Ebola Virus Disease
J. Casals : You have mentioned that there were difficulties with the neutra lization test with Ebola virus, we know that this happens with Lassa. What the type of difficulty that you have ?
G. van der Groen : The difficulty with the neutralization test is that were unable to produce plaques with Ebola virus, so we had to neutralize CPE in Vero cells. However, the virus in the tubes with the neutralization mix breaks through 1 or 2 days after CPE appears in the controls. Perhaps complement was to be added, but this remains to be done.
J.G. Breman : You haven't said it but I suppose your interpretation is that there are false positive serologic results. Of the 38 convalescents 20 had illness, 4 had no illness and no contact with patients, the other 14 had contact but no illness. None of the titers of less than 32 anywhere had symptoms nor contact. What exactly does the positives in the San Blas Indians at a titer of 64 mean ? How does this affect future epidemiologic studies ? As I recall, Dr. Casals screened some of the people that had recovered from Lassa fever against a number of different antigens and did not find any Oro reactivity with Lassa fever. I don't know if that is applicable here but it may be something that could be done in the future as long as we rely on the FA-test.
G. van der Groen : I think that is a priority now, we have so many serologic results and we must confirm these by a method completely independent of thisne. How to interpret the high positives, or the 1/4, 1 have no idea. In Zaire we found 5 positives, one with a high titer, in a village outside the epidemic area. These perhaps were asymptomatic infections. The lower titers could be the result of an epidemic that occurred in the past, although we do not know how long antibodies remain in the circulation. Dr. Simpson told us about the rapid decrease of antibodies in some of the people he investigated. We had the same phenomenon with a plasma donor; nine months after the acute diseases : practically undetectable. This man gave a lot of plasma, how much this influenced his antibody titer I do not know.
K.M. Johnson : it is becoming clear, to us at least, that the more work you do with the FA-test the more interesting, the more complicated and the more biologically sloppy the results become. I would urge very great caution in making any kind of final interpretation of what you have just heard. My own guess is that most of the reactions that are 64 or greater, really do represent specificity. I cannot explain how a Panamanian Indian can have antibodies to Ebola virus. I don't think these are real antibodies. Of course if these are not, it means that any others in a given serum may not be as well. It is clear that we must have an alternative and a much more specific method with which we can answer these questions. Several facts suggest endemicity of Ebola in Zaire. Of the five individuals who were bled and found positive at 1/64 or greater and had never any contact with the hospital or with a fatal case, four were found again six, seven months later and rebled. They were all still positive. Titers here and there might be twofold lower but they were all still positive. So at least we measured the right people the first time. Secondly of a group of people, largely plasma donors, rebled at the same time, in other words anywhere from about seven to eight months after illness, only one now has apparently no detectable antibody, and I'm not yet sure whether we got the right specimen. I'm beginning to believe that the virus may in fact be endemic in Zaire.