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ISOLATION, MONITORING AND TREATMENT OF A CASE OF EBOLA VIRUS INFECTION

RONALD T.D. EMOND
The Royal Free Hospital, Infectious Diseases Department, Coppetts Wood, Muswell Hill, London N10 1JN England.

In the second half of 1976 specimens from a serious outbreak of haemorrhagic fever in Zaire and the Sudan 1 were sent to highsecurity laboratories in Belgium, England and the United States of America where a distinctive virus was isolated which was subsequently designated Ebola . On 5 November 1976 one of the investigators at the Microbiological Research Establishment in England, accidentally pricked his thumb while transferring homogenised liver from a guinea-pig infected with this new virus5. In accordance with standard safety protocol he immediately removed the glove and immersed his thumb in hypochlorite solution then squeezed it tightly. There was no bleeding and careful examination with a hand lens failed to reveal a puncture mark. He was kept under surveillance and on the sixth day became ill.

Shortly after midnight on 11 November his temperature rose to 37.4ºC. During the early morning he complained of nausea and central abdominal pain but there was no headache or myalgia. About 14 hours after onset he was seen at the Microbiological Research Establishment, where a blood sample was taken for direct electron microscopy and guinea-pig inoculation. He was then transferred to the high-security infectious diseases unit at Coppetts Wood Hospital in London and admitted directly into a Trexler negativepressure plastic isolator(6,7).

On arrival he felt physically exhausted and complained of anorexia, nausea and constant central abdominal pain. There were no other symptoms. His temperature was 38ºC with a relative bradycardia. He was alert and did not seem to be particularly ill. Apart from slight abdominal tenderness there were no other abnormal findings. Since it appeared highly probable that the illness was due to infection with Ebola virus, treatment was started that same evening, 20 hours after onset of symptoms, with human interferon, which had been prePared by stimulating peripheral lymphocytes with Sendai virus Interferon was given by intramuscular injection in a dose of 3 million units every 12 hours for 14 days.

The following morning his temperature had returned to normal and he was free from symptoms, but later in the evening his temperature rose again to 39º C.

His appetite remained poor but no other symptoms developed. By this time direct electron microscopy of his blood had revealed Ebolalike virus particles. In view of this finding it was thought advisable to give the patient convalescent serum obtained from people who had recovered from the illness in Africa. Treatment of this serum to ensure safety presented serious problems. The closely related Marburg virus has been shown to persist in the body for several months after the acute illness, though it has not been shown in the circulating blood. Marburg virus is relatively resistant to heat but is inactivated in serum maintained at 60ºC for 60 minutes9,,* The Ebola convalescent serum was therefore treated at this temperature for 60 minutes to ensure safety. The serum was also tested for HBs Ag and HBs Ab because carriers are common in many parts of tropical Africa. 450 ml serum was given by slow intravenous infusion over a period of four hours from 1.30 a.m. on 13 November, commencing 47 hours after onset of illness. Blood samples were taken at frequent intervals to ascertain virus and antibody levels.

There was no obvious change in the clinical condition of the patient until the fourth day of illness, 14 November, when an erythematous maculo-papular rash was noted over the chest wall. About mid-day he had a sudden violent bout of shivering followed by a sharp rise in temperature to 40oC. This was accompanied by nausea, retching and a single episode of vomiting. Since admission he had been constipated but at this point he had a loose bowel action. His mental state began to change and over the next 24 hours there was striking deterioration in concentration and memory. Protein was detected in his urine and persisted thereafter until the fever subsided. Over the next 72 hours, when the fever was at its height, there was severe malaise and extreme weakness. Profuse watery diarrhoea developed and continued for two days accompanied by persistent vomiting. The rash spread to all parts of his body and ultimately became confluent. There was no bleeding into the skin or mucous membranes. The throat was inflamed and a few small patches of thrush were detected. The abdomen was slightly distended but there was no tenderness or guarding. He was mildly dehydrated and urinary output was falling. Metoclopramide was prescribed for the vomiting and Lomotil for the diarrhoea.

On the sixth day of illness, 16 November, a further 330 ml of convalescent serum, pretreated in the same manner, was infused and followed by Hartmann's solution to correct dehydration. Next day his urinary output fell to its lowest volume of 830 ml despite adequate fluid replacement and a satisfactory blood pressure. At this point his appetite began to return; vomiting and diarrhoea became less frequent and ceased after the 18 November. Swallowing proved painful and examination showed extensive candidiasis of the throat, which responded to treatment with amphotericin B lozenges. The erythematous stage of the rash began to fade on 19 November leaving staining over the limbs on the same day he complained of stiffness of the small joints of his hands and to a lesser degree of the wrists and knees. The oliguria and proteinuria present at the height of the illness could have been attributed to deposition of immune complexes in the kidney, especially in view of the transient arthral gia at the end of the acute stage, but these features were recorded in severe cases during the original Marburg outbreak, when no serum was given.

After 20 November his general condition improved. His fever subsided to a low level, his energy began to return, and there was dramatic improvement in his interest and ability to concentrate, though he could barely recollect the acute phase of his illness. The joint symptoms did not persist. The temperature returned to normal on 22 November but there was a further flicker of fever on the next two days after which the temperature remained normal. Output of urine was normal by 23 November. Subsequently he made an uneventful but slow recovery over 10 weeks. At the end of the acute stage he had lost a considerable amount of weight which he regained slowly during convalescence. The rate of growth of hair slowed during the acute illness and during convalescent cence there was considerable loss of hair from his scalp. There were no other clinical complications.

In the early stage of the illness facilities were not available for conducting haematological or biochemical studies safely, so efforts were concentrated on establishing the virological diagnosis; in the late stage of the illness when provision had been made for routine tests, they were not required for the management of the patient, though they proved useful for assessing the extent of the damage during convalescence(10). Fortunately, there was no bleeding and the use of prophylactic heparin was not considered to be necessary. Electrocardiograms taken during the acute stage were normal though the amplitudes of the T-waves were lower than in a recording made on the 27 January during convalescence. Blood urea, and sugar concentrations and liver function were normal during convalescence. The HBs Ag and HBs Ab tests on blood were negative. The result of a chest radiograph was normal. During the early period of convalescence the haemoglobin level and white blood cell counts were depressed and did not fully recover until 8 February 1977, three months after the onset of illness. Bone marrow depression was shown during the original outbreak of Marburg disease and was attributed to the activity of the virus. interferon also causes bone marrow depression affecting the stem cells of the granulocytes(11-13) and synthesis of haemoglobin(14). Furthermore, interferon causes immunodepression(15) and may have contributed to the severity of the thrush. Once the haemoglobin and white blood cell levels had returned to normal the patient was subjected to plasmaphoresis and a total of seven units of plasma were taken between 16 and 25 February 1977.

It is not possible to assess the value of interferon and convalescent serum from experience with one patient. While the course of the illness was milder than expected from reports elsewhere, the pattern and duration of symptoms were not modified. Although there was no obvious clinical improvement after treatment, there was a striking fall in the level of circulating virus. On the first day of illness a blood sample was found to contain 10(4.5) guinea-pig infective units/ml; on the day after starting treatment with interferon there was no change in the amount of virus, but on the next day after the infusion of serum, the level in the blood dropped to 3-10 guinea-pig infective units/ml and remained at this level until the viraemia disappeared on the ninth day of illness, before the temperature had returned to normal. The second infusion of serum had no effect on the amount of virus. Since there is known to be a time lag before interferon produces an effect on virus levels it is not possible to assess the relative effectiveness of the two preparations in clearing the blood.

Before the infusion of serum the fluorescent antibody titre in the patient's blood was 1/2; after the infusion of 450 ml convalescent serum with a fluorescent antibody titre of 1/128-1/256, circulating antibody was detected in the patient's blood at a titre of 1/16. This was consistent with the dilution of the convalescent serum. Circulating antibody remained at this level until the tenth day when the titre increased to 1/32 and gradually rose to a maximum titre of 1/128 by day 34. After plasmaphoresis the level dropped to 1/32 and fell further to a titre of 1/16 on 5 May 1977.

The patient was nursed in a Trexler negative-pressure plastic isolator within a high-security section of the Hospital throughout the acute stage of his illness and during convalescence until certain clearance tests proved to be negative for Ebola virus. Air pressure within the isolator was maintained below atmospheric and extracted air was drawn through a HEPA (high efficiency particle arrester) filter before being discharged above roof level. All supplies were introduced through an entry port without breaking the air seal. Infected material was removed in a similar manner into plastic bags which were sealed to prevent contamination of the surroundings. Dry waste was destroyed by incineration within the high-security area; liquid waste was pretreated with 1% Hycolin, a cresolic disinfectant, before being boiled. Doctors and nurses had access to the patient but were separated physically by a plastic film barrier.

Once the acute stage had subsided it was decided to take specimens for clearance tests at weekly intervals and it was arbitrarily agreed that three negative sets of cultures from throat swabs, blood, urine and faeces would be an acceptable standard for discharging the patient from isolation. After two sets of specimens had been shown to be free from virus the patient was removed from the isolator and transferred to a high-security room equipped with airfiltration and facilities for the safe disposal of excreta. He remained there pending the results of the third set of clearance specimens. Altogether he had spent 32 days in the isolator.

The contents of the isolator were removed and destroyed by incineration or packed for autoclaving. The room and the interior of the isolator were then fumigated with formaldehyde and left for 24 hours, after which the canopies were dismantled and destroyed by burning. The room was refumigated with formaldehyde and sealed for 24 hours. The staff undertaking these tasks wore full protective clothing and biological respirators.

When the tests of the third set of specimens proved to be negative the patient was discharged home. In view of previous experience in Germany with Marburg virus(16), a sample of semen was taken on day 39 and found to contain 3-10 guinea-pig infective units/ml. However this discovery was not thought to justify further isolation, especially as the patient fully appreciated the implications. Semen was positive again on day 61 but negative on days 76, 92 and 110.

The Trexler negative-pressure plastic isolator and the techniques used for the disposal of waste proved to be effective in preventing spread of Ebola virus from the patient to attendant staff and to the general community. Of the 24 nurses who were directly concerned in the care of the patient, six became ill with acute respiratory infections, which lasted on average two days. Four of the five doctors looking after the patient developed a 'flu-like' illness with some gastrointestinal symptoms. At onset these illnesses caused concern but the problems invariably resolved within two or three days and antibody studies later showed no evidence of Ebola virus infection among either medical or nursing staff.

REFERENCES
1. Weekly Epidemiological Record (1976) 51, 325.
2. Johnson, K.M. et al. (1977) Lancet, 1, 569.
3. Bowen, E.T.W. et al. (1977) Lancet, 1, 571.
4. Pattyn, S. et al. (1977) Lancet, 1, 573.
5. Emond, R.T.D. et al. (1977) British Medical Journal, 2, 541.
6. Emond, R.T.D. (1976) Postgraduate Medical Journal, 52, 563.
7. Emond, R.T.D. (1977) British Medical Journal, 2, 559.
8. British Medical Journal (1976) 1, 64.
9. Bowen, E.T.W. (1969) British Journal of Experimental Pathology, 50, 400.
10. Rutter, D.A. (1977) British Medical Journal, 2, 24.
11. Fleming, W.A. et al. (1973) Immunology, 23, 429.
12. Nissen, C. et al. (1977) Lancet, 1, 203.
13. McNeill, T.A., Gresser, 1. (1973) Nature, New Biology, 244 (11), 173.
14. Falcoff, E. et al. (1973) Journal of Virology, 12, 421.
15. Johnson, H.M. et al. (1975) Journal of Immunology, 114, 403,
16. Martini, G.A. (1973) Postgraduate Medical Journal, 49, 542.
DISCUSSION
A.W. Woodruff : None of the speakers have mentioned any deafness, as has been reported in some cases of Lassa fever; this could be a clinical difference between Lassa and Marburg-Ebola. Secondly, has one of them noticed in the pharynx a membrane which could be confused with a diphteritic membrane, as we observed in one of our patients with Lassa in London ?
M. Isaacson : Personally I have seen one case, but although she had very swollen tissues and a rather dirty-looking pharynx, I would not have confused it with a diphteritic membrane. The sore throat was very intense, the patient did complain also of the feeling of a painful lump in the throat, in fact it was subjectively the most worrying feature in this patient.
P. Piot : Deafness was not observed but tinitus was, and this may indicate involvement of the nervous system. We saw one patient with a greyish patchy exsudate on the soft and hard pallates but there was no membrane.
T.E. Woodward : My experience years ago was with patients with Korean Epidemic Hemorrhagic fever. I was struck by certain similarities and I want to ask about the blood pressure. I saw that the pulse became rapid in almost all patients, while myocarditis was mentioned by all speakers. In 1952 in Korea, it was noticed that if too much fluid is given it can be bad rather than good. In fact there was absolutely no integrity of the vascular system and fluid actual ly leaked into the tissues including the myocardium. It seemed that the careful titration of epinephrin did maintain the circulation for a while, but too much epinephrin was bad, too much fluid was bad and even if more than two units of serum albumin were given in an attempt to bolster the circulation, that too would pour too much fluid into the vascular system. I just wondered about how well these patients tolerated intravenous fluid ?
N. Isaacson : Case no 3 that we treated in Kinshasa, as I mentioned, did exhibit oedema: her face was oedematous, her upper extremities were oedematous, how much of this was due to fluid retention or to cardiac failure we don't really know. The renal output seemed to be reasonable. We were faced in Kinshasa, as well as in all the other parts where the disease occurred, with really a lack of laboratory facilities. This is perhaps one of the problems that has become most prominent for all of us who had to deal with it. We had patients there who needed both isolation plus sophisticated care with all the facilities that are required and we just did not have them. With a situation of intervascular coagulation and of possible impending cardiac failure, of possible myocarditis, we required electrocardiography, we required anticoagulation facilities, we required all the necessary Laboratory facilities and we did not have them. This made our work extremely difficult, I don't think this patient got adequate care. The blood pressure remained normal throughout.
L. Eyckmans : It is clear from the presentations of this morning that the hemorrhagic component is a very bad prognostic sign, but was actual blood loss contributive to death or was it only a bad symptom ?
M. Isaacson : At least in one of our cases I think blood loss was a feature. Our last cases had limited blood loss, nothing very serious.
D.P. Francis : There is a tremendous amount of intratissular and diarrheal loss though, and some of the patients, especially those with diarrhoea, appeared just like cholera patients with deep-set eyes and the typical skin. There is a lot of fluid loss.
F. Dekking : Dr Emond, how do you disinfect the isolator after use ?
R.T.D. Emond : We spray the inside of the isolator with 1% hypochlorite solution, Leave it twenty-four hours, then wash it out thoroughly and put the isolator back into use. If the patient had a dangerous infection, we destroy it, and the only occasion on which we actually did this was in the particular patient described. We sealed the room where the isolator was, we fumigated the room with formaldehyde generated by heat, put heat generators also inside the isolator and ran the pumps so that formaldehyde was drawn through the filters. We left it all for twenty-four hours and then wearing protective clothing and respirators we dismantled the canopy. The canopy was in fact too large to go through the opening of any of the incinerators we had, so rather primitively, we dug a hole in the field and burned it. I cannot think of any safer way of disposing of it. The filters were dismantled, sealed, autoclaved, incinerated and replaced by fresh ones. This is a technique we have used, and I'm willing to change my way if someone can suggest anything better.
M. Isaacson: What would be wrong with disinfecting the isolator for example with ethylene oxide and re-use the canopy ?
R.T.D. Emond : The total cost for this particular episode was so enormous, that the canopy was negligeable in it. If you consider that all the scientific staff in Porten were put off work and under surveillance, that our hospital with 160 people was put out of action, and that a great many community phi,,,--' cians were involved, the total cost must have run into 100.000 or 200.000 pounds. The price of the envelope is about 900 pounds Sterling.
M. Dietrich : Did you ever consider to use peracetic acid ? What was done with the waste coming out the isolator ?
R.T.D. Emond : Peracetic acid is used for the disinfection of these envelopes, The problem with it is that it is unstable, it has to be freshly prepared and we have preferred to use hypochlorite for ordinary routine purposes. I would not be prepared to put a fresh patient into an envelope in which we had nursed a patient with an Ebola, Marburg or Lassa virus infection. I think it is much wiser to destroy these envelopes. All dry waste was stored in plastic bottles inside the isolator until the bottles were full, treated with disinfectant for twenty-four hours, then removed in sealed bags and disposed of by boiling.
K.M. Johnson : Nobody asked what the action mechanism of passive antibody in a systematic disease like this could be. Why was it deemed necessary, even in the beginning to quarantine medical staff that was protected by the bed isolator ?
R.T.D. Emond : In Great Britain as in other countries, there was considerable at the reports which were coming from Africa about this new disease. The equipment at that time, although it had been used on a considerable number of occasions, had never been used in any really serious infection. Thirdly, one of the children of the patient developed a mild fever and it was thought it was possible that it was going to spread within the family grouping. Taking all things into consideration, it was thought advisable that the staff should be asked to go into voluntary quarantine which they agreed to do.

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