Racebrook Country Club, 7 MAI 1974
To Jean-Pierre Digoutte with compliments of Sonja M. Buckley
"This way it was."
To begin with, I would like to thank you for inviting me to be your guest speaker. Of course, my two colleagues, Drs . Casals and Downs, very kindly allowed me to come in their place - in this time of woman's liberation. I surmise that some of you have read the book condensation of ''Fever !'' in the March issue of Reader's Digest. But I hope that many of you will enjoy reading the book. At the beginning of April, I was asked to review this documentary for the American Society of Microbiologists. Although I was familiar with the Lassa fever story, once the book was in my hands, I could not put it down. It is suspenseful and reads like a medical detective story.
To-day, I would like to discuss two aspects with you: 1) how did the Lassa fever episode in 1969 affect our personal and private lives? ; and how do we look at the Lassa fever problem in 1974 ?
I come to you from the Yale Arbovirus Research Unit where we do usually work with arboviruses. This unit is under the directorship of Dr. Robert Ellis Shope. It is like a small department within the Department of Epidemiology and Public Health. The latter is chaired by Dr. Robert Wayne McCollum. It was Dr. McCollum, the chairman , who allowed us to work with the Lassa fever specimens in 1969. Arboviruses are transmitted by insects such as mosquitoes, and ticks. I am sure that you are all familiar with mosquitoes, but for those of you who have never seen a tick, I'm wearing to-day a small silver broche which contains a tick. Now, if we ask ourselves very briefly, what has happened in arbovirus research during the last 20 years? About 300 different arboviruses have been recognized. At the present time, we at the Yale Arbovirus Research Unit have well over 300' virusea stored away in our deep freezers; and we also have inmune sera prepared against all of these viruses. What is more important, we have experts such as doctors Anderson, Casals, Clarke, Downs, Karabatsos, Shope and Tignor in our unit who can identify viruses. These experts can also diagnose diseases serologically. Some of the staff members such as doctors Aitken and Wallis grow these viruses in mosquitoes. And some staff members such as doctor Karabatsos and myself have a great deal of experience in growing these viruses in tissue culture. So, in a way, it was only logical that the Lassa fever specimens of the three American nurses landed in our unit in I969, because we have the tools to track down strange infectious agents.
What exactly were Dr. Casals and I doing when Dr. Downs brought the specimens from the nurses? On that Thursday, March 6th, we were working with tick- borne viruses; for instance, Colorado tick fever virus is a tick-borne virus. Both of us had been invited to take part in an international symposium on tick-borne viruses. It was going to be held in Bratislava, Czechoslovakia. Dr . Casals was classifying about 20 of these viruses and I was growing them in different cell lines - I had cell lines derived from mosquitoes, from baby hamsters and from Indian and African monkeys. What was our attitude towards these Lassa fever specimens. They were at that time unwelcome intruders. We were not afraid, but we were pessimistic. He knew that Lilly Pinneo, the head nurse, had been stationed in the Pest House in Lagos for 4 days. We surmised that the blood specimens collected from Laura Wine and Charlotte Shaw had probably been thawed and frozen many times - after all, Laura Wine had died in January and Charlotte Shaw in February. However, we had hoped to isolate some agent from the critically ill Lily Pinneo, now hospicalized at the Columbia Presbyterian Hospital. On that Thursday , March 6th , I called my pathologist husband and I told him that I would be delayed. Doctor Downs arrived with a thermos bottle filled with specimens at 4:00 PM in the afternoon. We had a conference on the 7th floor in Dr. Casals' s section. The meeting pertained to procedure and strategy . What to do? Dr. Downs said : ''He have to inoculate mice (baby mice are the choice host system for isolation of arboviruses) - but the diseaae is not typical for arboviruses - so, we better inoculate also some tissue cuItures. I mentioned that I did not have one single spam culture left. Then, Dr . Casals turned to me and said : ''Sonja, be sure to take all the time you need and prepare your cultures well.'' This I did on the very next day with Elinor Gilson, my technician. In what kind of a mental from was I? I really had no hope of isolating anything. But I had great pride in our unit. I remembered that Dr. Downs had told me that the very same specimens had also been submitted to three* other laboratories. So, the element of competition was there. On that Friday , we prepared 240 stationary tube cultures and 70 plaque bottles, all together 310 cell cultures. What cell lines did I select? Based, in my experience, I knew that arboviruses liked baby hamster kidney cells and also Indian kidney cells. But in the past I had also worked with viruses causing South American hemorrhagic fevers. I remembered that these viruses liked African green monkey kidney cells; that latter cell line is called the Vero cell line (Vero in Esperanto language means '' thruth''). While Elinor and I were busy in the sterile room, Dr . Downs came into my section. He looked like a surgeon, dressed with gown, mask and rubber gloves. He carried a rack with many glass tubes. The test tubes were carefully dated and labeled with names of the nurses. I removed small amounts from the tubes, just enough to inoculate my cultures. I had no gown, mask or gloves. My long-time and trusted technician, Charles Mullen, froze the samples down in our deep-freeze. At the end of the day, Dr. Casals came into my section - actually, my section adjoins his - and looked with satisfaction into my large walk-in incubator where all the cultures were growing. After the weekend, Charles Mullen and I spent the whole day on Monday with the inoculation of the cell cultures. Every time we would leave the sterile room , we would waah our hands. We carried all the contaminated pipettes and other glassware to the autoclave ourselves. These precautions were used throughout the whole year of 1969. Also, I assigned three special rooms in my section to the work with Lassa fever specimens. During the next following days, nothing exciting happened - the mice remained healthy, the cultures remained beautiful and the results of the serological tests were all negative. We aid : ''A whole week wasted!'' We departed !or the weekend - Dr . Casals to New York and I to Stamford.
On Monday, I had to take a day off. 0n Tuesday morning, I was so disinterested in the Lassa fever experiments that I did not bother to check the cultures. Then around 2: 00 PM in the afternoon, Dr. Max Theiler, a Nobel prize winner and my former director, came up to my laboratory for coffee. He asked me : ''Do you have anything new?'' And I said: ''Oh, I have not even checked the cultures yet. '' Right there and then, I started to examine my cultures under a large Zeiss microscope which I have in my office. I examined the inoculated Vero cultures first. To my great astonishment, every single inoculated Vero cell culture : degenerated, dying cells; the control cultures looked beautiful. I went to Dr. Theiler and told him that the specimens of Laura Wine, Charlotte Shaw and Lilly Pinneo had destroyed the Vero cells. He said in a very low voice: ''African hemorrhagic fever !'' I begged Dr. Theiler not to tell Dr. Downs who was at that time in his office on the 6th floor. But within the hour, Dr. Downs was in my office where I was still examining cultures. He said : ''I hear you have aomething ! '' I answered: ''Yes, but I have to make a second passage to be sure.'' He agreed. I also showed the damaged cells to Dr. Casals. The fhree of us were puzzled by the fact that the tissue cultures were more sensitive than the mice. I went home that night, excited, but tired and my husband cooked the meal, as he was going to do many times during 1969. One week later, all the second passages were down. I knew at that time that I had definitely isolated an agent. How did this affect my life? In the laboratory, whenever I was working on the Lassa experiment, I slowed down and became meticulous. Inasmuch as possible, I remained on my own floor and started to avoid people a little bit. At home, I told my neighbors to please keep their children out of our yard. I stopped entertaining completely. In the laboratory, I made a third passage with a strain isolated from Lilly Pinneo, fixed the dying cells with Bouin's fixative and gave this preparation to my husband. In his hospital, he made a Giemsa stain and called me: ''Microscopically , this looks very much like a virus you have been working on before'' he said to me. 0n the next day, I looked at the preparation myself and indeed, it looked like Junin virus, the :etiologic agent of Argentinian hemorrhagic fever. Many people have asked me later : ''Here you not terribly afraid to work with Lassa virus?'' I can say that I was very apprehensive until I saw the Giemsa preparation; but as soon as I saw it, I had confidence that I could handle the agent.
In the meantime, the nurse Lilly Pinneo was on the way to recovery. I could not detect any more virus in blood specimens which had been taken on March 20th and March 29th. As a matter of fact, Dr. Casals found complement fixing antibodies and I found neutraLizing antibodies in the blood specimen 29 May. That is to say when I mixed the virus isolated from Lilly Pinneo early in March with her serum obtained on March 29th, and when I inoculated this mixture into new Vero cells, the cells remained healthy and beautiful and did not die. Dr. Casals also could show by complement fixation test that the three viruses isolated from the three nurses were identical. This meant that the virus from Laura Vine had infected Charlotte Shaw, and the virus from Charlotte Shaw infected the head nurse Lilly Pinneo. I believe that the index case, that is the case which infected Laura Vine, was never located. At this point, the agent was baptized ''Lassa virus'' and we started to characterize it and classify it. In these studies, we were assisted by doctors Glarke, Shope, Speir, Wood and Liebhaber. And it was determined by serology and electron microscopy that Lassa virus was indeed like a first cousin to the viruses causing the South American hemorrhagic fevers. At this point, we relaxed a little. At least, we had pin-pointed the enemy down. At the end of May - ''May 29th to be exact - Dr. Casals asked me if I had any objection to the inoculation of baby and adult mice with Lassa virus tissue culture material? Of course not. I gave him some material and he inoculated the mice in a special, large sterile room which is located right next to his office. Jimmy Washington, Dr . Casals's technician, took care of the mice over the long Memorial Day weekend. Early in June, Dr. Casals came to me and was quite excited. He said: ''All the baby mice are still fine, but most of the inoculated adult mice have died; the few still living look like they have a disease caused in mice by lymphocytic choriomeningitis virus!'' I went with him and stood under the door of the sterile room. Dr. Casals removed an adult mouse from its cage , spun it by its tail whereupon the mouse started to have convulsions, stopped breathing temporarily and had rigidly extended hindlegs. As he explained to me: ''These signs closely resemble those seen in adult mice inoculated with lymphocytic choriomeningitis virus'' - I discreetly stepped :way from him. I remembered my Swiss microbiology teacher, Professor Mooser, famous for his typhus work. He used to say: ''You don' t have to be afraid of a corpse; but be aware of any living thing which sneezes, coaghs, urinates or defacates. '' As I'm talking to you right now, it comes to my mind that this adult mouse possibly urinated while Dr. Casals spun its tail. Anyhow, exactly six days later, on June 10th, Dr. Casals was ill with a cold, and in addition, he must have had chills, because he was wearing a thick gray sweater underneath his white laboratory coat. During this week, he came in every day. His cold got worse. On Friday, he was pale white and was complaining of having severe leg pains. It so happened that Mrs . Casals and their daughter Christina had been visiting the grandmother in Long Island; when they returned on Sunday evening and saw Dr. Casals, he looked like a very sick man - they were very alarmed and especially Christina insisted on calling the family physician vho arranged for immediate admission to the Columbia Presbyterian Hospital. I learned of this only Monday, June 16th, when Dr. Downs called a staff meeting. We knew at that time that our friend had a severe illness, characterized maihly by high fever, a low white cell count, pharyngitis and unusually severe muscle pains in the legs. We all suspected Lassa fever. Especially Dr. Downs wanted to initiate treatment with immune plasma from the nurse Lilly Pinneo right away, but the physician in charge would not hear of this. He wanted the diagnosis ''Lassa fever'' established first. Dr. Downs arranged for a messenger service of specimens from Dr. Casals, and Jimmy Washington would usually bring a blood specimens from Dr. Casals, but also throat washings and urine specimen; H: This was done on Monday, Tuesday and Wednesday, But the tissue culture isolations would take at least 4 days and in the meantime, Dr. Casals went down-hill rapidly. On Wednesday morning, we had another staff meeting in the library on the 6th floor. This meeting was attended by Dr. McCollum, our chairman, and by Dr. Dorothy Horstmann, professor of epidemiology and public health. We were all in favor of giving the immune plasma. Lilly Pinneo came down to New York by plane and a drop infusion of immune plasma was started Wednesday evening and continued during the night. Jimmy Washington continued to bring specimens and to my great delight, the blood specimen collected two days after the administration of immune plasma was free of virus and all the blood specimens collected thereafter were clean. Dr. Casals started to recover, but he cantinued to be contageous, because he was shedding virus in the throat washings and in the urine. However, by the middle of July, all laboratory reports on urine and throat specimens showed negative findings. To our great joy and relief, Dr. Casals was ready to go home. He was saved specifically by administration of antibody derived from Lilly Pinneo.
Little did we know at that time that five years later Dr. Casals was going to save the life of a German physician, Dr. B. Mandrella, ill with Lassa fever. And this brings me to the latest Lassa fever episode during February and March of 1974. It occurred in Onitsha, East Central State, Nigeria. The sequence of events went more or less as fol1ows: at the request of the Center for Disease Control (C.D.C .), Atlanta, Georgia, Dr. Casals was assigned by he Rockefeller Foundation to join a team sent to Nigeria to investigate this latest outbreak of Lassa fever. He was accompanied by Dr. Downs. The two Yale professors of epidemioLogy left on March 16th. Very briefly, this is what they found : a possible or suspect index case (confirmed since by CF test, CDC, Atlanta, Georgia), a young Nigerian male, was admitted to the St. Charles Borromeo Hospital, Onitsha, with malaise, temperature of 100-101° F and a few vague complaints. He was examined by Dr. Sauerwald, a 30 year old German national on the hospital staff. Mrs. Sauerwald was also in Onitsha and they were expecting their first child. The young Nigerian man was released from the hospital after two days and later readmitted with fever and a psychotic behaviour. He was again examined by Dr. Sauerwald. About nine days later, Dr. Sauerwald, the first contact case, came down with typical Lassa fever. When a respiratory obstruction developed, a tracheotomy was performed by Dr. B. Mandrella. Dr. Sauerwald died the same day after convulsions and deep coma. Dr. Mandrella became ill with fever 7 days later. He also experienced malaise, muscle pains and sore throat. He did not respond to treatment with antibiotics and antimalarial drugs. When there was no improvement, Dr. Mandrella was sent to Ibadan, Nigeria. Most important at this time is the fact that a certain Dr. David Toms, a Canadian physician, was considering the possibility of new Lassa fever outbreak. As my Swiss professor of medicine, Dr. Loeffler, used to say; "A good diagnostician is a doctor who thinks of the right diagnosis at the right time. "This is what Dr. Tons did. And he wasted no time and contacted a Dr. White in Jos who came down to Ibadan and examinated Dr. Handrella. Dr. White had treated many Lassa fever cases during the 1970 outbreak in Jos. Dr. White made the diagnosia of Lassa fever. Dr Mandrella was given immediately two units 'of Dr. Casals's immune plasma and recovered thereafter. In the meantime, Lassa virus has been isolated from both Drs. Sauerwald and Mandrella. Dr. Mandrella was then transferred to Germany on a special plane supplied by the German government. Dr. Casals went directly from Nigeria to the headquarter of the World Healtn Organization at Geneva, Switzerland. Some of the chief niedical officers, there, are very much concerned with the problems created by transport of patients vith Lassa fever from one country to another where there is no Lassa virus as yet.
As a matter of fact they scheduled right there and then an informal conference on Lassa fever to be held on May 2-3 in Geneva, Switzerland. Dr. Casals has been attending this conference and has just returned. He reported to us Yesterday informally during a staff meeting. As regards transportation of people suspected of having Lassa fever, the following was recommended: if a Lassa fever patient wants to go to a certain nation, that nation has to supply the plane and isolation facilities. As regards isolation of Lassa fever patients, the following was recommended: patient should stay in isolation until patient ceases to shed virus from urine or throat washings; there should be repeated negative laboratory reports, possibly at least three negative reports. Specifically, these experts also recommended the development of a vaccine; methods for early diagnosis and ecological studies such as distribution of the rat Mastomys natalensis and also study of its microhabitat. A document pertaining to these recommendations will be prepared and given to the Director General who in turn will present the document to the Assembly (made up of representatives of countries participating in WHO). This Assembly will meet within the week.