Ebola Hemorrhagic Fever Outbreaks in Gabon, 1994–1997: Epidemiologic and Health Control Issues
- Alain-Jean Georges,
- Eric M. Leroy,
- André A. Renaut,
- Carol Tevi Benissan,
- René J. Nabias,
- Minh Trinh Ngoc,
- Paul I. Obiang,
- J. P. M. Lepage*,
- Eric J. Bertherat*,
- David D. Bénoni,
- E. Jean Wickings,
- Jacques P. Amblard*,
- Joseph M. Lansoud-Soukate,
- J. M. Milleliri,
- Sylvain Baize and
- Marie-Claude Georges-Courbot*
- Centre International de Recherches Médicales de Franceville, Franceville, Ministère de la Santé Publique, Faculté de Médecine, Université Omar Bongo, Mission Française de Cooperation et d'Action Culturelle, and Ministère de la Santé Publique, Libreville, Gabon
- Reprints or correspondence (current affiliation): Dr. Alain-Jean Georges, Chefferie, Hopital d'Instruction des Armées Desgenettes, 108 Bd Pinel, 69 275 Lyon, Cedex 03, France (ajgeorges{at}wanadoo.fr).
Abstract
From the end of 1994 to the beginning of 1995, 49 patients with hemorrhagic symptoms were hospitalized in the Makokou General Hospital in northeastern Gabon. Yellow fever (YF) virus was first diagnosed in serum by use of polymerase chain reaction followed by blotting, and a vaccination campaign was immediately instituted. The epidemic, known as the fall 1994 epidemic, ended 6 weeks later. However, some aspects of this epidemic were atypical of YF infection, so a retrospective check for other etiologic agents was undertaken. Ebola (EBO) virus was found to be present concomitantly with YF virus in the epidemic. Two other epidemics (spring and fall 1996) occurred in the same province. GP and L genes of EBO virus isolates from all three epidemics were partially sequenced, which showed a difference of <0.1% in the base pairs. Sequencing also showed that all isolates were very similar to subtype Zaire EBO virus isolates from the Democratic Republic of the Congo.
Johnson et al. [1] isolated and identified Ebola (EBO) virusas from human cases during a 1976 epidemic of hemorrhagic fever (HF) in the Democratic Republic of the Congo (DRC). During the same year, EBO virus was isolated from patients during an HF epidemic in Sudan [2]. The viruses, which were closely related to Marburg virus (all members of the Filoviridae), had an 88% and a 53% case fatality rate in DRC [3] and Sudan [2], respectively. A third outbreak, with a case fatality of 60%, occurred in Sudan in 1979 [4]. In addition, a death was registered in Tandala, DRC, in 1979 [5]. In 1994, a new strain of EBO virus was isolated from a Swiss researcher with a dengue-like syndrome, who had likely been infected during the necropsy of a chimpanzee (see Formenty et al., this supplement). The animal had been found dead in the Taï National Forest (Côte d'Ivoire) during a 2-year epidemic that killed half of the population of chimpanzees [6]. A third serious human epidemic of EBO occurred in 1995 in Kikwit, DRC; it was associated with a mortality rate similar to that seen during the DRC and Sudan epidemics [7].
During 1994 and 1995, an outbreak of HF occurred in northeastern Gabon. It was first considered to be caused only by yellow fever (YF) virus on the basis of the clinical symptomatology, routine biochemical tests, and specific laboratory results provided by the Centre National de Référence des Fièvres Hemorragiques Virales (Institut Pasteur, Paris) [8, 9]. However, retrospective serologic tests detected concomitant EBO virus antibodies among some of the patients and the general population [10, 11]. Later efforts to isolate the EBO virus from some of the specimens from the first epidemic (1994) were successful. One year later, in February and in July 1996, two more HF outbreaks occurred in northeastern Gabon [10–13].
Herein, we report on three EBO epidemics that occurred between late 1994 and early 1997 in northeastern Gabon [10].
The Epidemics
It is important to note that during this investigation, we continually faced many difficulties (e.g., logistics problems and cultural and political constraints) in the collection of data and management of our research on this disease. Despite our efforts, the difficulties sometimes led to the loss of important information, and at times, forced us to report the scientific data in a rather unorthodox manner.
First epidemic (fall 1994)
The first epidemic in Gabon had two waves of patients, with the first beginning in early December 1994 and the second beginning at the end of January to February 1995. All patients in the first wave came from 3 gold-panning encampments (Mékouka, Andock, and Minkébé) situated in small clearings of 2000–3000 m2 at the edge of the rain forest. Figure 1 and table 1 show the geographic location of these and other villages with case-patients. Table 1 also shows the chronology of illness for the cases. Three hundred fifty people, mainly of the Bakota ethnic group but also some Bakwélé, inhabit this area (30 in Mékouka, 20 in Andock, and 300 in Minkébé).
Figure 1
Geographic location of sites of primary and secondary cases of hemorrhagic fever. A, Gabon, B, Africa, C, sites.
Figure 2
Evolution of 1994–1995 Ebola virus epidemic in Gabon. A, Evolution by mortality rate. Curve shows biphasic evolution of epidemic limited to large no. of secondary cases who have spread virus. B, Geographic evolution of epidemic. Hop = hospital.
Figure 3
Evolution of spring 1996 Ebola virus epidemic in Gabon. A, Evolution of epidemic by mortality rate. Curve shows monophasic evolution of epidemic limited to small no. of secondary cases who spread virus. B, Geographic evolution of epidemic. Not included is case who died on 19 February 1996 and who had different infection not related to Ebola virus since 20 January 1996. Hop = hospital.
Table 1
Geographic location (in order of occurrence of illness) of sites of primary and secondary human Ebola infections during the fall 1994 outbreak in Gabon.
A total of 32 sick persons from the three forest encampments (23 from Mékouka, 4 from Andock, and 5 from Minkébé) traveled 100 km south by river to the nearest hospital at Makokou, the main town of the region, for treatment. In addition to the primary cases in Andock, we were also informed that deaths had occurred in the local population of great apes (chimpanzees, Pan p. troglodytes, and gorillas, Gorilla g. gorilla). However, despite intensive searches of the forest by teams from Centre International de Recherches Médicales de Franceville (CIRMF), no cadavers or skeletons were ever found, even when a patient told us that he had killed a chimpanzee with abnormal behavior inside his encampment. This report could be anecdotal, but it cannot be totally ignored.
The second wave of patients did not originate from the encampments: They were what we should have called (in a survey following accepted procedures) secondary (or tertiary ?) cases. Unfortunately, the information that was available from the authorities regarding these patients did not provide a precise genealogy of all cases. The first patient in the second wave was from Mayela, a small village close to Makokou, far from the forest encampments. This person, who was probably the first secondary or tertiary case, lived near a traditional healer (a “nganga”) and was probably infected as a result of contact with a hospitalized patient who, against medical advice, left the hospital to seek care from the nganga. Sixteen more cases (table 1) occurred in mid January: 1 case at Makokou Général Hospital, 12 cases from Mayela, and 3 cases from two villages (Ekataniabé, 1 patient; Ekobakoba, 2 patients) on the road running south toward Franceville. None of these patients had been in the area affected by the first wave of the epidemic during the previous 3 months; however, all had been either in direct contact with sick relatives (people hospitalized at Makokou Général Hospital or sleeping in the nganga traditional healer's home) or with people caring for patients.
The last reported case (infected while caring for a relative at Makokou's hospital) occurred at Ekobakoba on 9 February 1995, and the epidemic was declared over by Gabonese health authorities on 17 February 1995.
Overall, 49 persons were admitted to Makokou's hospital with suspected HF. Patients were suspected of having HF if they had a well-identified contact with an ill person or at least two of the following clinical signs: fever, diarrhea, vomiting, melena, conjunctivitis, arthralgia or myalgia, diarrhea, or vomiting. Of the first 9 patients, 2 who we examined during the first wave also had jaundice, which has never been described as a symptom of EBO infection but is consistent with YF infection. This case definition of HF, which was proposed by the physicians in charge of the Makokou hospital (a very poorly equipped facility), was rather broad, and at least 4 of the 49 patients appeared unlikely to have HF. On 18 December 1994, during the middle of the first wave, the Gabonese health authorities requested that we examine 9 patients in Makokou General Hospital. Biologic samples from those patients led to the identification of the etiologic agents.
Between December 1994 and March 1995, CIRMF collected 22 samples in Mayela from contacts (all ⩾15 years old) of sick persons and 88 samples from the local population of Ogooué-Ivindo Province, Gabon, where the epidemic occurred. At that time, many people had fled the area in terror; therefore, it was difficult to conduct an investigation. A year later (January 1996), we sampled 236 people from the three initially infected villages and from villages in the Makokou area in order to further assess exposure to EBO virus in the local population.
Second epidemic (spring 1996)
A second epidemic began during early February 1996 in the village of Mayibout 2, Gabon, which is located on the Ivindo River. Mayibout 2 is 40 km south (6 h by boat) of Mékouka and Andock, where the first epidemic broke out, and north of Makokou (7 h by boat). Eighteen people who had skinned and chopped a chimpanzee cadaver that they found became ill (fever, headache, bloody diarrhea). They were evacuated from Mayibout 2 to Makokou on the decision of the village chief, despite governmental instructions to the contrary. All patients were admitted to Makokou Général Hospital, where 4 moribund patients died within 48 h.
The bodies of the 4 patients were returned by river to Mayibout 2; a fifth patient, who was moribund when he escaped from the hospital, died after returning to Mayibout 2. Traditional burial ceremonies were performed without any special precautions to avoid disease transmission. Two other “primary cases” occurred, which appeared not to be connected to the chimpanzee episode; 1 died. Fifteen serum samples from these initial 18 primary cases and 6 from secondary and tertiary cases were obtained. An additional 205 serum samples were obtained from the population of Mayibout 2 and neighboring villages (Mayibout 1 and Mvadi, which are 2 km south and 20 km north, respectively, of Mayibout 2).
Third epidemic (fall 1996)
On 5 October 1996, we informed the Gabonese health authorities (who 1 week earlier had requested our assistance in the investigation) that we had isolated EBO viruses from 2 of 6 samples from patients hospitalized at Booué. Personnel from CIRMF carried out a retrospective investigation of this third epidemic, which probably started as early as 13 July with the death (undeclared) of a 39-year-old hunter in a logging camp near Booué (0°06′ S, 11°57′ E) between Ovan and Koumameyong, 200 km from Mékouka and 120 km from Makokou to the southwest (figure 1). The symptoms of this first case were suggestive of viral HF (VHF; fever, bleeding, vomiting, diarrhea, and headache). At the beginning of August 1996, information was obtained that several chimpanzees may have died in the same area, and a field collaborator was able to obtain skin samples from 1 cadaver for pathologic examination at the Centers for Disease Control and Prevention (CDC, Atlanta), with which CIRMF was in contact.
Six weeks later, at the end of August, a second hunter (35 years old) died in the same Gabonese logging camp, but his death, which was also suggestive of VHF, was not linked to that of the first hunter. A third hunter (26 years old) became ill 12 days later and was hospitalized at Booué. He subsequently evaded the medical authorities at Booué and died in the village of Balimba, where he was being treated by a nganga. Shortly after the hunter's death, both the nganga and his nephew fell ill in mid-September and were admitted first to Booué Hospital and then transferred to Makokou General Hospital, where they died. Three other cases, including another nganga, occurred among people living in the same area.
After we confirmed that the patients had EBO HF (EHF), the authorities decided to apply the same clinical and epidemiologic definitions that were used during the outbreaks in Yambuku, DRC, in 1976 and in Kikwit to diagnose EHF patients [1, 7]. The disease spread around Booué; 24 cases, including 17 deaths, were officially declared by 13 November 1996. A Gabonese doctor, who had performed an endoscopy in a private hospital in Libreville on an EBO-infected patient from Booué, developed signs and symptoms of EHF. On 27 October, without first requesting a serologic diagnosis of VHF and without considering the possible etiology of such a diagnosis, he went to Johannesburg for treatment. A South African nurse who cared for the doctor became ill on 2 November and died on 24 November (Swanepoel R, personal communication).
At the end of November 1996, a second wave of this third epidemic appeared in three locations around Booué: Lolo (3 deaths, 6 cases), SHM, a timber company (4 deaths, 5 cases), and the logging camp of Balimba (1 death, 1 case). A confirmed case spread to Lastourville (130 km southeast of Booué) from Balimba, and again to Libreville, with subsequent transmission of the disease to the capital (11 deaths, 15 cases).
The epidemic was declared over in March 1997 (last officially declared patient: 18 January), with a total of 60 cases and 45 deaths. Unfortunately, the information made available to us from the local authorities did not allow for a very precise genealogy of all cases; however, we did obtain accurate data on 47 of the 60 cases (Milleliri JM, unpublished data).
Materials and Methods
Routine Clinical Chemistry Tests
Routine clinical chemistry tests were done on sera from some hospitalized cases in each epidemic.
Serologic Assays
IFAs
IFAs were used to detect viruses causing HF (including Crimean-Congo HF fever virus, Rift Valley fever virus, subtype Zaire of EBO virus [EBO-Z; Mayinga and Boniface strains], Lassa fever virus, and Marburg virus) in sera from some patients in each epidemic. The IFAs were done using CRELM (initials of each virus) microscope slides [14] provided by the CDC and US Army Medical Research Institute of Infectious Diseases (Fort Detrick, Frederick, MD).
IgM anti-EBO EIA
ELISAs were also used to detect EBO virus infection [15]. For IgM detection, an immunocapture test was used. Polyvinyl chloride 96-well microtiter plates (no. 1000-3, Bioblok; Dynatech, Strasbourg, France) were adsorbed overnight at 4°C, using goat anti-human m-chain (Tago, Burlingame, CA) diluted 1:500 in 0.01 M PBS (pH 7.4). All steps of the assay were done using volumes of 100 µL/well. Sera from persons who were infected or were suspected of being infected and sera from their contacts were added to the wells at a 1:100 dilution in serdil (5% skim milk [Bacto milk], PBS, 0.1% Tween 20). After the plates were washed with PBS with 0.1% Tween 20 (Bio-Rad, Richmond, CA), sera were added (first at a 1:100 dilution and then at 4- to 800-fold dilutions), and the plates were incubated for 60 min at 37°C in a humid chamber. For each serum and each dilution, in one well, we used as antigen a cell slurry made in EBO-infected Vero E6 cells diluted 1:1000 in PBS; in another well, we used a slurry of uninfected Vero E6 cells. Anti-EBO (subtypes Z, Sudan [S], and Reston [R]) hyperimmune rabbit serum was diluted 1:2000 in serdil. Plates were incubated for 1 h with anti-rabbit IgG (no. 074–1506; Kirkegaard & Perry, Gaithersburg, MD) diluted 1:10,000 in serdil (as conjugate) and then incubated for 30 min using an H2O2− ABTS substrate (Kirkegaard & Perry).
In the first assays we used 2 standard serum samples as positive controls. As soon as we had confirmed the EBO cases, we added our own 2 positive controls, which were run in standard dilution series for providing standard curves, which allowed the determination of the limits of detection of the assay. A panel of 4 negative sera (from healthy persons) were run in all tests to determine the background of the assay (the limit at which the positive controls are positive) and to provide the mean and standard deviation (SD) of that background. A sample was considered to be positive if its optical density exceeded the mean + 3 SD of the controls. Plates were read spectrophotometrically at an optical density of 410 nm on a microcomputer (LP 200; Sanofi Diagnostics Pasteur, Marnesla-Coquette, France).
IgG anti-EBO EIA
For IgG detection, half of a 96-well microtitration plate was coated with a lysate of EBO-Z—, EBO-S—, and EBO-R—infected Vero E6 cells, and the other half was coated with a lysate of uninfected Vero E6 cells to determine the specific binding of antibody to virus antigens (the background); both lysates were diluted 1:1000 in PBS. Plates were incubated overnight at 4°C. The plates were washed three times with PBS plus 0.1% (vol/vol) Tween 20, and then sera were added at a 1:100 and 4- to 800-fold dilutions in serdil and allowed to react with the antigencoated wells for 60 min at 37°C in a humid chamber. Bound IgG was detected with mouse anti-human IgG conjugated to horseradish peroxidase. Further identification was done as in the IgM EIA assay. A panel of specific IgG-positive controls and specific IgG-negative sera (from healthy persons) was run in all tests in order to determine the cutoff value. For each assay, the mean and SD of the adjusted optical density were accumulated and used to calculate a value equal to the mean + 3 SD, which represented the cutoff value. Plates were read as above.
Anti-YF IgM EIA
At the beginning of the first VHF epidemic, YF virus was strongly thought to be the probable cause of illness, on the basis of the clinical and epidemiologic results of the 9 patients examined. Therefore, we tested for YF virus infection, using IgM capture techniques [17].
Virus Isolation Attempts
Vero E6 cell 6-well plates were inoculated with sera from patients suspected of harboring a VHF virus. During the first epidemic, the biosafety level 4 facilities of the Institut Pasteur were used for all virologic manipulations of patient-derived material; further work on the two last epidemics was carried out at CIRMF's site or at CDC's Special Pathogens laboratories or at both sites for part of the molecular biology testing of EBO virus strains [16].
To identify the EBO virus, EBO reference antisera were tested against the virus isolates cultured on Vero E6 cells. Tests were done using polyclonal antibodies raised in mice (IFA test) and a pool of monoclonal antibodies (antigen-capture test) that were known from previous experience to identify EBO antigen for different strains of virus in tissues. Molecular identification of the EBO virus strains following extraction of RNA from whole blood clots or from the peripheral blood mononuclear cells separated on ficoll have been described elsewhere [16].
At Institut Pasteur, attempts to isolate YF virus by inoculation of suckling mice, mosquitoes, and mosquito cell cultures (AP 61, C6–36) failed to give positive results. Therefore, RNA was extracted according to the method of Chomczynski and Sacchi [18], and the presence of the YF viral genomic RNA was revealed by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification [19] using consensus primers for flaviviruses. The amplified DNA fragment was then transferred onto a nitrocellulose membrane and hybridized with a YF virus—specific probe.
Antigenemia
The sera of sick people, their contacts, and people suspected of having EHF were tested by EIA for the presence of EBO antigen, as were the supernatants of Vero E6 cells seeded with sera from these persons. Half of a 96-well microtitration plate was coated (overnight at 4°C) with a mouse monoclonal antibody recognizing EBO virus epitopes; the other half was coated with normal myeloma ascites diluted 1:1000 in PBS. Biologic samples to be checked for the presence of EBO antigen were then added (first at a dilution of 1:4 and then 4- to 64-fold). After a 1-h incubation at 37°C in a humid chamber, the plates were washed 3 times, and rabbit anti-EBO (subtypes Z, S, and R) diluted 1:2000 in serdil was added and incubated for 1 h at 37°C in a humid chamber. Plates were incubated for 1 h with anti-rabbit IgG conjugate (no. 074–1506; Kirkegaard & Perry) diluted 1:10,000 in serdil and then incubated for 30 min using an H2O2− ABTS substrate (Kirkegaard & Perry), and bound rabbit IgG was detected.
Results
Serologic surveys
The occurrence of HF cases in the fall of 1994 initially led us to suspect an outbreak of sylvatic YF infection. However, we were aware that another HF agent (in particular EBO virus) could be circulating, given the possible fatalities observed in nonhuman primates (apes replicate YF virus, but they usually do not die from it; Monath TP, personal communication).
In the first instance, we screened 9 patients (all from Makokou General Hospital) for HF virus. EBO IFA on CRELM slides was negative for all 9 serum samples, whereas 6 of 9 patients showed evidence of recent YF virus infection (IgM capture). Of the 49 sick patients, another 14 cases who had never been vaccinated against YF were also screened. Of the 14 cases, 8 had evidence of recent YF infection as determined by IgM capture or Southern blot, but none had evidence of antibodies to the main African viral HFs as determined by use of IFA on CRELM slides. However, the antigen used in our IFA test was several years old, so in June and July 1995 we used ELISAs and fresh antigens to retrospectively test these same 23 samples (ELISA is generally more sensitive than IFA). Nineteen patients had signs of a recent infection with EBO as determined on the basis of at least one of the following: positive for EBO IgM, positive for antigenemia, positive for virus isolated from cultured Vero E6 cells.
We also used ELISA to test the 88 serum samples obtained in January 1995 (before YF vaccination) from the population living around the three encampments (Andock, Minkébé, and Mékouka) where the first epidemic broke out and the 22 serum samples obtained from contacts in Mayela. Two of these sera (1.8%) were positive for anti-EBO IgG but not EBO IgM, while 9 YF-unvaccinated people (8.1%) were positive by YF IgM capture, indicating a recent YF virus circulation.
One year later, we obtained samples from 236 persons from the same area (table 1). For various reasons, only 56 of the original 110 people were part of this second cohort (e.g., use of given name in the place of family name in original survey, fear of being identified and accused of causing the original epidemic, and migrations of populations). Of 236 subjects, 24 (9.7%) had anti-EBO IgG by ELISA without any anti-EBO IgM (1 of these IgG-positive cases was the first case of the first wave in the 1994 epidemic and had survived a typical EBO-type illness).
During the second epidemic, 205 samples were collected from people living in 3 villages (Mayibout 1 and 2, where EBO was known to have occurred, and Mvadi, where 1 unconfirmed death of a hunter was said to have occurred). High frequencies of anti-EBO IgG (14.9%–30%) and IgM (5.7%–10.0%) by ELISA in all three villages (table 2) may suggest the presence of an asymptomatic or mild form of the disease concomitant with the classical severe form of EBO, or they may demonstrate the limits, in terms of specificity, of the EBO IgG and IgM ELISAs that we used.
Table 2
Anti-Ebola antibodies among the general population of three villages in Ogooué-Ivindo Province, Gabon, during the second epidemic (spring 1996), as determined by ELISA.
During the initial phases of the third epidemic, we were unable to carry out serologic surveys due to local reasons that were out of our control.
Fatality and descriptive epidemiology
We reviewed files at Makokou General Hospital for persons who were seen during the first epidemic in fall 1994; 49 patients were identified with at least two signs of VHF, 29 of whom died (case fatality rate, 59%). There were 26 male and 23 female patients (sex ratio, 1.1) with a mean age of 37 years.
For the spring 1996 epidemic, the case fatality rate was 67.7% (21 of 31 cases died) as determined using the accepted (Yambuku and Kikwit) case definition. There were 17 male and 14 female patients (sex ratio, 1.2) with a mean age of 27.6 years.
In the fall 1996 epidemic, there were 60 cases and 45 deaths (case fatality rate, 75%). The sex ratio was 2.4. We were unable to conduct adequate surveys during the epidemic, so complete age-range data are unavailable.
The development (geographically and chronologically) of the first two epidemics is shown in table 1 and figures 2 and 3, respectively. When tracing the computerized (MS Access software; Microsoft, Redmond, WA) polynomial regression curves obtained from histograms, the first epidemic shows two waves of morbidity, whereas in the second outbreak, there is only one peak of morbidity. These observations are related to the notably different types of spread in the two epidemics. Persons in several villages were infected during the first epidemic, which lasted longer than the second, in which only persons in a confined area were affected. The exact origin(s) of the virus in the first epidemic is uncertain, and despite reports of the death of great apes, we were unable to confirm these reports.
Exploitation of the area by gold miners has caused substantial disturbance to the forest canopy. This disturbance may have caused contact between other species and humans that otherwise would not have occurred. None of the specimens (small mammals and insects) collected in the field so far have yielded any sign of harboring the virus; however, these studies are ongoing.
In the second outbreak, there is no doubt that the virus came from one source, a chimpanzee infected by EBO (for which the chain of infection is unknown). The chimpanzee seems to have been the index case for infecting 18 primary human cases. There were 2 other primary cases, for whom we have no information on the source of infection (the role of a nganga was suspected), making a total of 20 primary cases for this epidemic. Only those people who had been in contact with the dead chimpanzee before it was cooked for eating were affected; nobody was infected by eating the cooked meat. We identified 10 secondary cases involving close contacts of dead or dying people and 1 tertiary case. It is important to note that no cases of infection occurred among the professional medical personnel because of preventive measures that were set in place on the second day of the epidemic. All equipment and material necessary for barrier nursing and prevention of the spread of disease were provided. One hundred ninety-one contacts were traced, with no evidence of infection by EBO virus.
Similar to the case in the first epidemic, the source(s) of the virus in the third epidemic was not immediately obvious, nor could it be traced retrospectively. The lack of identification of cases at the outset and the lack of adequate containment measures once the diagnosis had been made meant that sick persons could travel throughout and even outside Gabon. Some contact cases could not be identified and isolated.
Immunostaining [15] done at the CDC on skin biopsies from a chimpanzee found dead in the forest indicated the presence of EBO [16]; however, the ape is very unlikely to have had any contact with humans.
Biologic and clinical features
Clinical signs and symptoms observed in 15 serologically or virologically confirmed patients from the outbreak at Mayibout 2 (spring 1996) are presented in table 3. Fever was always present, while in ∼75% of the cases, diarrhea (a dense suspension of black or brown-red uneven microaggregates <5 mm in size in liquid or syrupy stools) and vomiting were seen, and ∼50% had conjunctivitis or conjunctival bleeding. Taking only the second epidemic into consideration, since it was the best-documented outbreak, clinical signs and symptoms were observed between days 6 and 11 after handling the chimpanzee, and deaths occurred between days 12 and 18 after the appearance of signs or symptoms. Despite a small number of observations, it seemed to us that those who died had a shorter incubation period than those who survived.
Table 3
Clinical manifestations observed in 15 confirmed cases of Ebola hemorrhagic fever during the spring 1996 outbreak in Mayibout 1 and Mayibout 2, Gabon.
Table 4 presents the biologic and biochemical data for 13 patients infected during the spring 1996 outbreak. Of 13 patients in the cohort, 6 died. Data were obtained 4–7 days after the onset of disease (16 days after the patients were infected by a chimpanzee cadaver). The levels of direct and conjugated bilirubin were normal, but all patients had signs of liver involvement (i.e., elevations in γ-glutamyltransferase, amino-transferase, and alkaline phosphatase), and 23% had signs of slightly decreased renal dysfunction (elevated serum creatinine and urea).
Table 4
Biochemical results for 13 patients with Ebola hemorrhagic fever during the spring 1996 outbreak in Gabon.
Most of the other blood parameters (e.g., total serum proteins, albumin, uric acid, cholesterol, triglycerides, sodium, potassium, and chloride) that we were able to test were normal.
Characterization of virus strains
EBO viruses were isolated from patients from each of the three epidemics. All the Gabonese virus strains sequenced showed a high level of homology with the group of strains from DRC [16]. Viruses isolated from patients during the same outbreak had identical base-pair sequences, but base-pair sequences were slightly different for isolates from the individual outbreaks. There was only a four-nucleotide (nt) difference in the GP gene in each of the 3 Gabonese strains (i.e., the GP gene from the December 1994 strain differs from that of the February 1996 strain by four nt, and the February strain differs from the October 1996 strain by 4 nt).
A conserved region of the L gene was also amplified in virus strains from fall 1994 and fall 1996, using degenerate primers selected from sequences of EBO-S and Marburg virus strains; PCR products were subsequently sequenced [13]. The 2 Gabonese strains showed high homology in the first 156-bp sequence, with only a single base change (position 110; A to G) separating the 2 strains. As stated, during the first epidemic, 6 of 9 patients had anti-YF IgM. At the Institut Pasteur, using PCR and specific hybridization techniques, YF sequences were identified in sera from 2 of the 6 patients who were anti-YF IgM positive and in 1 of the 3 patients who had no anti-YF IgM. Those 3 patients, who were negative for all EBO investigations, had clinical signs compatible with YF (e.g., severe low back pain, fever, jaundice [2/3 patients], melena, and melanemesis), and biochemical results showed signs of liver and renal failure and slightly raised bilirubin levels.
Discussion
An epidemic of HF among humans, some of whom had clinical and biologic symptoms suggestive of YF infection, started in December 1994 in Ogooué-Ivindo Province. On the basis of clinical, biologic, and serologic data, a diagnosis of YF infection was suspected in 6 hospitalized cases, and a diagnosis of YF infection was confirmed for 3 other cases on the basis of molecular biology data, although the complete virus could not be isolated, which is not unusual for YF virus. (YF virus is known to be impossible or difficult to isolate from clinical material if samples are taken several days after evolution of the clinical disease; for example, during the 1970 epidemic in Okwooga and the 1986 epidemic in Oju region, Nigeria, it was impossible and difficult, respectively, to isolate YF virus [Monath TP, personal communication]). At the time, no etiologic diagnosis was scientifically established for the 6 patients who were not confirmed to have YF virus infection; nevertheless, we felt, from a public health point of view, that there was a risk for reemergence of YF (the last epidemic in Gabon was in 1949 [8]) among the unvaccinated population, as confirmed later by the epidemiologic investigations of a World Health Organization expert [9].
Without waiting for a definitive diagnosis, for which investigations were in progress at the hemorrhagic fever unit at the Institut Pasteur, and despite the fact that another etiologic agent could be involved, CIRMF suggested to the Gabonese health authorities that they rapidly instigate measures to limit the spread of YF infection. We suggested (1) passive isolation of infected case-patients, with barrier nursing, disinfection, and insect eradication programs and (2) active reintroduction of a vaccination program against YF virus, which had lapsed >10 years earlier. The fact that the entire YF virus was not isolated is not a conclusive argument against the fact that YF occurred during the first VHF epidemic, since samples may have been collected at a late stage of the disease when the virus is difficult to isolate. However, in fall 1994, there was a high prevalence of anti-YF IgM in YF-unvaccinated subjects, which was strongly suggestive of a recent YF outbreak. It is known that sylvatic YF infection can exist in a mild or symptom-free form [17].
A final argument for the presence of YF virus during the first epidemic (fall 1994) comes from Pisano et al. [20], who identified YF infection in a cohort of 5 patients (different from ours) from the same area by sequencing a cDNA fragment obtained by RT-PCR and showing 94.4% homology with the Asibi strain of YF virus [21].
During the first epidemic, EBO and YF viruses were obviously coexisting: 9 patients had EBO alone, 11 had EBO and YF, and 3 had YF alone. Due to the small size of each group, it is impossible to speculate on the relative lethality of each virus alone or in combination, but a nosocomial amplification of EBO appears to have occurred inside Makokou Général Hospital. Such an association of YF and EBO viruses during the same epidemic has been suspected in the past: In Ethiopia, where a VHF outbreak occurred in 1961–1962, a later serologic retrospective study showed that both EBO and YF viruses may have been present [22].
Earlier IFA-based studies looking for the presence of EBO virus in other provinces of Gabon were done by Ivanoff et al. [23] in 1982 and Meunier et al. in 1986 [24]. Data obtained by use of the same technique showed that EBO is widespread throughout equatorial Africa [25–27]. However, these results must be examined carefully since others have suggested that IFA is likely to give false-positive results [28]. In addition, intrain our hands, this test was insensitive for EBO virus, specifically during the fall 1994 epidemic, in which all sera collected during the acute phase of the disease tested negative by IFA, while some were positive by EIA. Even though ELISA techniques appear to be very sensitive for assessing EBO infection, we must be cautious about their specificity.
No serologic data were available from persons in Ogooué-Ivindo Province before the first epidemic. However, we had kept 58 untested frozen serum samples from the epidemic period. The samples were from asymptomatic adults who were not associated with gold-panning; 2 of them had ELISA EBO IgM, suggesting recent EBO infection, and the remaining 56 samples were negative for anti-EBO IgG and IgM. This low percentage of seropositivity suggests that the circulation of EBO is a new phenomenon in the area, affecting mainly people living deep in the forest in temporary encampments.
There are interesting demographic and ecologic differences between the sites of the first two epidemics. The gold panners in the fall 1994 outbreak were a transient population and caused considerable damage to the local forest in which they were working. Most of those who survived left the area after the epidemic; a very small population remains. Mayibout 2, a permanent, relatively dense settlement whose inhabitants practice slash- and- burn cultivation, is situated on the river Ivindo and is surrounded by forest. The disturbance of the forest integrity is one common feature between the two sites, but the one important difference is the permanence and the size of the population around Mayibout 2, which might permit the virus, once present in the area, to circulate within the human population. Both the reservoir and the vector for the EBO virus are still unknown; however, it is apparent that the great apes, or at least chimpanzees, are not involved because they are as susceptible as humans to the disease.
On the basis of a sizeable but incomplete genetic comparison, the viruses from our three outbreaks are essentially indistinguishable from the viruses isolated in DRC in 1976 and 1995; however, the viruses isolated in 1976 from Sudan and DRC were clearly biologically distinct from one another [29]. We cannot rule out the possibility that varied strains of EBO virus or even different members of the Filoviridae are circulating in these areas, with some being more virulent than others yet cross-reacting and, therefore, appearing as asymptomatic infections in the population. The inability to isolate viruses from the Gabonese environment and from anyone but ill patients has not allowed us to address this issue. The fact that the ELISA test has not been evaluated for specificity against known human infections also does not permit us to eliminate this as a possible explanation for the high level of antibody in the population of Mayibout 2.
Finally, it is very clear that the use of simple barrier nursing methods in even the most basic of hospital settings is sufficient to limit the spread of this disease. Nosocomial spread of infection due to the lack of recognition of the disease and the dearth of training and material available to establish simple barrier nursing remains the root cause of most of the disease observed to date. From the public health point of view, this remains a basic goal for the prevention of EHF epidemics. Preventing primary infection certainly requires the identification of risk factors and the reservoir, something that continues to elude us all.
Acknowledgments
We thank B. Le Guenno (Institut Pasteur, Paris) for his invaluable scientific assistance in affirming the yellow fever diagnosis in January 1995 and for initiating new retrospective serologic studies on Ebola virus in June 1995 and new attempts at virus isolation in samples from the fall 1994 epidemic, which led to the isolation of the first Ebola isolate from Gabon; P. Tshipamba (CIRMF) for providing excellent technical assistance; J. B. McCormick (Institut Pasteur), S. Fisher-Hoch-McCormick (Fondation Mérieux, Lyon, France), and P. Rollin (CDC, Atlanta) for valuable and pertinent suggestions regarding the manuscript; and members of the Special Pathogens Branch of the CDC as well as members of Walter Reid Army Institute of Research, US Army Medical Research Institute of Infectious Diseases (Fort Detrick, Frederick, MD), who have generously provided reagents to CIRMF since 1982.
Footnotes
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Informed consent was obtained from the patients or their parents or guardians.
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Financial support: CIRMF is supported by the Republic of Gabon, the French Ministry of Foreign Affairs (Coopération et Action Culturelle), and ELF GABON Co. Ltd. (Libreville).
- © 1999 by the Infectious Diseases Society of America
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