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Four Ebola subtypes are known to date: Ebola-Zaire, Ebola-Sudan, Ebola-Ivory Coast and Ebola-Reston. Infections with these viruses are rare, but they occupy a special place on account of their virulence and the media attention they receive. * Ebola-Zaire and Ebola-Sudan In 1976 there was a sudden large-scale epidemic of 2 different Ebola viruses in Maridi (South Sudan) and in Yambuku, on the Ebola river in North Congo. The mortality rate in Yambuku was very high (280 deaths out of 318 cases = 88%) and slightly lower in Sudan (53%). In 1977 there was one fatal case in Tandala, North Congo. New major outbreaks occurred in 1979 in Nzara (South Sudan), in 1995 in Kikwit, Congo and in 2003 in Kelle, Congo Brazzaville. The virus, which emerged in Kikwit, very closely resembled that in Yambuku (less than 1.6% difference in RNA, which is very little). This is a sign of a genome which is not under selection pressure, suggesting a stable ecological niche between epidemics. The Sudanese virus isolates of 1976 and 1979 were also almost identical. * In December 1994, 44 people were infected in Minkouka (northeast Gabon). In February 1996, 37 people were infected with Ebola virus in the isolated village of Mayibout, in the same area on the Ivindo river. In this case it was suspected that people were infected after eating infected chimpanzee meat. In October 1996 a similar outbreak was seen in Gabon in Booué and then in Makokou. There were approximately 60 cases. An infected doctor was flown over to South Africa and there caused a fatal secondary case in a nurse. This illustrates how easily pathogenic organisms can be spread in this age of rapid transport. The Gabonese virus isolates were identical to those in Congo. Asymptomatic infections occurred in Gabon in a number of people. In the autumn of 2000 there was a large scale epidemic of Ebola-Sudan in Gulu in the north of Uganda. After this there were cases in other districts (Masindi, Mbarara). There were 425 cases with 224 deaths. In December 2001-January 2002 numerous cases were reported in Gabon and in neighbouring Congo. Multiple cases emerged again in April 2002 in Gabon. Early in 2003, a large scale epidemic occurred in Mbomo and Kelle, a very remote and rural area of Congo Brazzaville, just south of Odzala National Park. It started by a large scale die-off among the lowland gorillas in the park. The disease flared up again in the same area, in November the same year, but was contained before New Year 2004. In May 2004, Ebola haemorrhagic fever appeared again in Sudan, in the area of Yambio. * Ebola Ivory Coast In 1994 many chimpanzees died following an Ebola epidemic in the Tai nature reserve in Côte d’Ivoire on the border with Liberia. Here one person was infected during an autopsy on a chimpanzee that had died. She was evacuated to Switzerland where she was treated. The causative agent turned out to be a new genetic subtype of Ebola virus. In late 1995 another (unconfirmed) case occurred in the same area (Plibo) in a Liberian refugee. *
Ebola-Reston In 1989 an epidemic of another Ebola virus occurred in a primate centre in the USA in Reston, a town near Washington D.C. A number of people were infected but without any illness both in Reston (4) and in the Philippines (12) where the monkeys came from. Unlike the case of Ebola-Zaire, there were arguments here for aerogenic transmission. Research was complicated by the fact that another haemorrhagic fever virus epidemic was taking place at the same time among the monkeys (Simian Haemorrhagic Fever Virus). The Ebola-Reston virus also emerged in 1992 in Siena, Italy. Again there was a connection with monkeys imported from the Philippines. In March and April 1996 too there were a few cases of Ebola-Reston in monkeys imported from the Philippines (Macaca fascicularis) in the Texas Primate Breeding Center, Alice, Texas. These animals were then still in the obligatory quarantine period (all monkeys imported into the USA have to be kept in isolation for a time after their arrival there). In January 1997 Ebola-Reston infections were confirmed in monkeys in the Ferlite Scientific Breeding Centre in the Philippines, the same centre where a similar outbreak had occurred in 1989. There were no human cases. * Summary of known human Ebola cases
3.2 Ebola virus, transmission
Transmission takes place through direct contact with infected body fluids (including sexual contact) and nosocomially through infected needles and contact with infected blood. Aerogenic transmission of Ebola has been demonstrated in the laboratory in Rhesus monkeys. The natural reservoir of these viruses is unknown. The monkey species which have been studied thus far all die from the infection and therefore cannot form the natural reservoir. Contact with infected monkeys plays a role in the beginning of an epidemic but how these animals are initially infected is not known. The epidemic which started in November 2003 in Mbomo, Congo Brazzaville, was rumoured to have started after villagers found a dead wild pig in the forest and ate its meat. This would be the first case that such an animal would be implicated. Certain fructivorous and insectivorous bats can be experimentally infected and certain species are seropositive in nature. These animals develop an asymptomatic infection. The viral genome has been detected by PCR in certain small rodents in the Central African Republic. These results could not be confirmed. Viral antigens could not be found nor was the virus ever cultured from these animals. The reliability of these PCR results is open to question. Structures, which may well have been viral nucleocapsids, were seen with the electron microscope in spleen cells in some animals. The animals belonged to two genera of rodents (Muridae; Mus setulosus and Praomys sp.) and one species of shrew (Soricidae; Sylvisorex ollula). Probably these leads were red herrings.
3.3 Ebola virus, clinical symptoms
People infected with Ebola virus suddenly experience fever, severe headache and muscle pain, malaise, extreme asthenia, conjunctivitis, occasionally a papular rash, dysphagia, nausea and vomiting, bloody diarrhoea followed by diffuse haemorrhages (particularly from the mucosa), delirium, shock and ARDS. Only 5% of the patients have jaundice. In addition to functional blood platelet disturbances there is always also thrombocytopenia. Initially there is lymphocytopenia and later neutrophilia. Histologically there is focal necrosis in various organs (testes, kidneys, liver, etc.). The haemorrhages, which may occur in such infections are partly caused by invasion of and damage to vascular endothelial cells. A component of the glycoprotein coat of the virus is toxic to vascular endothelial cells. The immunological course early in the infection determines how quickly the Ebola virus replicates and whether the host will die or recover. Surviving an infection is linked to an early appearance of IgM and IgG, followed by the activation of cytotoxic cells. People die if their humoral defence system is disturbed and T cell activation is too late to prevent virus replication. The role of several cytokines (IL-1 β, IL-6 and TNF) is being investigated. The production of these cytokines is stimulated among other things by activated endothelial cells. Asymptomatic infection with Ebola can occur. The virus isolated from these individuals is wild type rather than a less virulent variant.
3.4 Ebola virus, diagnosisDiagnosis during an epidemic is clinical or serological. It is significant that each of the various geographical isolates have their own antigenic structure and therefore problems can arise with serological testing, including Western Blot test. It is better if antigen can be demonstrated in blood or saliva (antigen capture ELISA) or the virus itself in tissue samples via electron microscopy. The viral RNA can be detected via a reverse transcriptase PCR (blood sample, mouth swab). Virus can be cultured in a few laboratories (e.g. on Vero cells). A new development is the use of immunohistochemistry, which can detect the virus in skin biopsy using monoclonal antibodies. A skin biopsy from a dead person can be kept in a simple but hermetically sealed container, in formalin, for later analysis. This avoids the difficulties, which are inherent in the use of a cold chain (liquid nitrogen, dry ice) needed for other diagnostic tests.
3.5 Ebola virus, treatmentThere is no known effective treatment. Ribavirin is not active in vitro. Administering blood from convalescent patients is controversial but was carried out to a limited extent in Kikwit. The use of equine hyperimmune serum has been studied in Russia. The use of recombinant antibodies against Ebola virus is experimental. The effect of steroids is being studied. In 1998 the first results of animal studies with a vaccine were published. It appeared possible to protect guinea pigs against Ebola virus by immunisation with plasmids coding for viral glycoproteins. (Guinea pigs are sensitive to Ebola). Data suggesting that extracts of Garcinia kola (bitter cola) contain substances that might possess anti-Ebola activity, are still to be confirmed. During epidemics, good nursing care might lower the mortality. * Infected macrophages display tissue factor, a clotting protein, on their surface (i.e. there is overexpression of this protein). When bound to factor VIIa, this activates the coagulation cascade, leading to localised blood clots. One hypothesis is that inhibition of the fVIIa/tissue complex could ameliorate Ebola symptoms. The anticoagulant rNAPc2 (recombinant Nematode Anticoagulant Protein; see ancylostomiasis) counteracts tissue factor. Treatment with rNAPc2 resulted in significant survival in Rhesus monkeys infected with Ebola virus. However, more study is needed to evaluate how this would apply in human infections.
3.6 Ebola virus, what to do in the event of an Ebola outbreak?
The very first step is to recognise possible clinical cases. Steps should be taken to identify and type the virus (send a blood sample safely to a well-equipped laboratory). In a laboratory which is protected and equipped to work with dangerous pathogens (so-called biosafety level 4), an attempt will be made to detect viral antigen, antibodies and viral RNA (reverse transcriptase PCR) and carry out an analysis of the genome in order to establish which Ebola subtype is involved. * If it is established that it really is Ebola, the government will be notified. Central control, registration and coordination is essential for combating an epidemic. Groups specifically responsible for a certain part of the campaign will be set up: clinical care, surveillance in the community, logistics, collecting the dead and safe burials, investigating rumours, informing the population, epidemiological study, research, reception centre, etc. These days it is also useful to appoint someone who can handle the press correctly. Every day information will be exchanged between the various teams and the latest developments will be reported to the WHO in Geneva. * The patients' movements should be limited. They should be isolated (no direct physical contact with patients, blood, excreta etc.). New patients must be directed to an emergency unit. Here, based on the history (contact with Ebola patients, fever, haemorrhagic symptoms) and a physical examination (melena, red eyes, haemorrhages), new patients must be divided into Ebola patients, non-Ebola patients and cases where there is doubt. Ebola cases must be kept in isolation. Cases where there is doubt should be kept elsewhere for observation (also in isolation). Often contact with Ebola will not be reported due to superstition, fear of stigmatization or if there was sexual contact with a person who subsequently developed Ebola infection. The absence of a rapid antigen detection test on-site can be a practical problem for the clinicians working in the field. * Personal protection (masks, goggles, aprons, boots, disinfection supplies) for medical staff and for people who care for the sick person (often family) is necessary. Demonstration of how to use the protective equipment and proper explanation are imperative. Equipment should be disinfected rigorously with, for example, bleach (hypochlorite solution). Objects, which cannot be sterilised, must be burnt under supervision. People who are suspected of being infected with the Ebola virus should be cared for by people who understand and use personal protection. Basic needs (drink, food, pain-relief, hygiene, etc.) have to be met. In emerging situations, when the medical staff is overwhelmed, only one member of the family should be allowed into the patient's room, and then only after thorough instructions and regular supervision. * Since there are no known active antivirals against Ebola virus, treatment will be symptomatic. In all epidemics so far, treatment was done in very basic circumstances and treatment in an intensive care unit was not possible. During epidemics, good nursing care might lower the mortality. Good hydration and nutrition is essential. High calory liquid food is easier to swallow that solid food, due to severe pain in the throat. Complications and other infections will be treated, e.g. metoclopramide (Primperan) or domperidon (Motilium) against vomiting, loperamide against diarrhoea, ranitidine against gastric bleeding. Paracetamol, ampicillin and quinine or another antimalarial will be given if bacterial surinfection and/or malaria are suspected. Chlorpromazine and even haloperidol might be considered in case of agitated confusion. Vomitus, sputum, faeces and urine will be collected in a plastic bucket and mixed with strong bleach before disposal. Aspirin is to be avoided. Rehydratation is important. * People who have recently had contact with Ebola patients but do not display symptoms have to be placed under supervision (surveillance) or in quarantine for 3 weeks. If this does not take place in a hospital, but at home, daily (radio) contact is desirable. * People who have survived Ebola have to remain in quarantine for a further 3 weeks after recovery. It is not known how long the virus continues to be excreted. Sexual transmission is possible up to 7 weeks after clinical recovery. Convalescent serum can be stored if necessary, but its use is controversial. Animal studies have been unable to prove any benefit of hyperimmune serum. * A general large-scale information campaign with adequate and practical information for the population should be started. If this results in many questions and tips, a permanent centre can be set up where information about possible new cases can be examined. In view of the extreme virulence, the incomplete knowledge about these pathogens and memories of the impact of the earlier plague and yellow fever epidemics, these pathogens can capture the imagination of the general public. Superstition and belief in witchcraft can lead to misunderstandings and violence. * Centres with a poor medical infrastructure and with a high risk of nosocomial transmission, have to be closed down temporarily. This applies both to large hospitals and small one-person clinics with only a few needles and syringes. Strict guidelines have to be issued to centres which continue functioning, particularly as regards disinfection, the use of needles and syringes, vaccinations and surgical procedures. In many places non-qualified private individuals have only a few (non-sterile) needles and syringes, which they use for all kinds of injections. * The deceased should not be washed and the bodies have to be isolated and buried as quickly as possible and reasonable. This sometimes causes problems with the family and acquaintances of the deceased because of the disruption of traditional rituals. The government has a role to play here in law enforcement. It is also useful to have an idea of the average mortality in an area prior to the Ebola outbreak. * Caring for orphans in the community should be organised if this does not take place through the traditional system of the extended family. The latter sometimes does not work because of fear, prejudice and practical problems. * Logistics play a very important role and include, among other things, equipment and materials, administrative support, accommodation, money and wages, communication, transport, fuel, safety and stock management. Good management is essential and has to be entrusted to reliable people. The NGO Médecins Sans Frontières has a lot of experience in handling the logistics of such operations. * Experts in various areas cannot, in most cases, make themselves available quickly for a long time and a rotation system should be organized. It is best if staff do not change too frequently in order to achieve a minimal continuity locally. Realistic guidelines for cases in which medical personnel are infected accidentally have to be drawn up. * Epidemiological research should attempt to identify transmission routes and secondary cases. One of the first questions which has to be answered is whether the course of the epidemic suggests aerogenic transmission or not. Risk factors for infection should be identified. An attempt will also be made to trace the first case in order to understand how the chain of infection started. However, this person may well have died. Several people, such as customers, work colleagues, neighbours, family and friends may be able to provide useful information. A reminder of the terminology: the index case is the patient in whom the disease first indicated the existence of an outbreak. The index patient always remains the same person irrespective of whether earlier cases are discovered later. The very first case is called the primary case, not the index case. Later secondary, tertiary, etc. cases can follow. * Because an animal reservoir is assumed, extensive attempts have been and are being made to identify this. An "ecological" team should be exclusively involved in this and will study different animals in the vicinity. An investigation should also be carried out into whether the virus is "exported" from the isolation units in the hospital to the environment. In addition to the fieldwork itself, there then follows the tedious analysis of the various potential hosts (both for the presence of the virus and their taxonomic identification). If new, more powerful and sensitive test methods are developed, samples from earlier field expeditions can, if necessary, be reexamined. A detailed description of the ecological/botanical environment in which the primary case emerged could be useful. As cases occurred in the Tai National Park in Côte d’Ivoire, where an ethological study of chimpanzees had been going on for years, this became a starting point for research. To date the analysis of the numerous arthropods and vertebrates has not produced a single positive viral isolate, but antibodies against Ebola virus have been found in fructivorous bats. It was discovered that the Ebola glycoproteins which make up the "shell" of the virus are chemically and structurally very similar to those found in several bird retroviruses. This suggests that birds might be implicated in the transmission of illness. At this moment, this is still hypothetical. * Rapid sample analysis (blood samples of patients, skin biopsies, etc.) and rapid transmission of the results is recommended. Logistical problems can hinder this. Investment in research and cooperation will pay dividends. * The maximum known incubation period is 21 days. After the end of the epidemic (no more cases for a minimum of 6 weeks), surveillance can be carried out locally. It is extremely likely that isolated cases and limited outbreaks will occur regularly. In order to obtain a better understanding of this disease, long-term surveillance is necessary. This can be done by analysing skin biopsies (immunohistochemistry) from deceased people who had suggestive symptoms before death. These biopsies could be performed and collected by locally trained doctors. The technique has the great advantages of being simple and having high sensitivity. No cold chain is necessary and transportation can take place safely. The sample is kept in a bottle of formalin. This kills the virus. The bottle is then immersed in a bleach solution in order to disinfect the exterior. The same technique is under evaluation for future monitoring of other haemorrhagic diseases with fever (e.g. yellow fever). * We do not know how all epidemics started, but several followed the consumption of infected apes. The risk of nosocomial transmission is clear. A safe medical infrastructure has to be built for the future. This is easier said than done. Naturally this does not only apply to Ebola, but to the whole spectrum of medicine. The cases of Ebola fever in South Africa and Switzerland show that the virus can emerge anywhere in the world, owing to modern rapid means of transportation. * At the end of 2000, the first favourable results of an experimental vaccine were reported in which cynomolgus monkeys (Macaca fascicularis) were protected against a fatal dose of Ebola virus, the Mayinga strain. An immunisation technique was used which was based on viral DNA injection followed by multiple boosters with a modified adenoviral vector. However, the vaccination regime took several months to produce immunity. In August 2003, Gary Nabel of the Vaccine Research Center at the National Institute of Allergy and Infectious Diseases in Bethesda, Maryland announced the results of a new trial. Cynomolgus monkeys were protected 28 days after a single injection of a modified adenovirus in which parts of the Ebola genome coding for nucleoprotein and glycoprotein were inserted. How long the protection lasts is not known at present and it is also not known if there is protection against other Ebola subtypes. If similar promising results with a one-shot vaccine would be obtained in humans, fast ring vaccination would become possible in case of a new outbreak. Adenoviruses are common human pathogens (e.g. common cold) and many people have anti-adenoviral antibodies in their blood. If this will interfere with this vaccination technique is not yet known. The use of alternative adenoviral serotypes might be useful if this would prove to be the case. Another vaccine is based on a modified animal pathogen, the vesicular stomatitis virus. When VSV's glycoprotein-gene was replaced by that of Ebola, the resulting virus protected animals against lethal Ebola challenge. However, VSV can occasionally provoke disease in humans, so there is some uncertainty about the safety of such an approach. So far, phase I studies are ongoing at the time of writing (March 2004).
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