j Infect Dis.-1999-Georges-s65-75 (Article Ebola Virus)

8/6/2019 j Infect Dis.-1999-Georges-s65-75 (Article Ebola Virus)

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S65

Ebola Hemorrhagic Fever Outbreaks in Gabon, 19941997: Epidemiologic andHealth Control Issues

Alain-Jean Georges, Eric M. Leroy, Andre A. Renaut, Centre International de Recherches Me dicales de Franceville, Franceville, Minist ere de la Sant e Publique, Faculte de Medecine,Carol Tevi Benissan, Rene J. Nabias, Minh Trinh Ngoc,

Universite Omar Bongo, Mission Franc aise de Cooperation et dActionPaul I. Obiang, J. P. M. Lepage,* Eric J. Bertherat,*Culturelle, and Ministe `re de la Sante Publique, Libreville, Gabon

David D. Benoni, E. Jean Wickings, Jacques P. Amblard,*Joseph M. Lansoud-Soukate, J. M. Milleliri, Sylvain Baize,and Marie-Claude Georges-Courbot*

From the end of 1994 to the beginning of 1995, 49 patients with hemorrhagic symptoms werehospitalized in the Makokou General Hospital in northeastern Gabon. Yellow fever (YF) virus wasrst diagnosed in serum by use of polymerase chain reaction followed by blotting, and a vaccinationcampaign was immediately instituted. The epidemic, known as the fall 1994 epidemic, ended 6weeks later. However, some aspects of this epidemic were atypical of YF infection, so a retrospectivecheck for other etiologic agents was undertaken. Ebola (EBO) virus was found to be present concomi-tantly with YF virus in the epidemic. Two other epidemics (spring and fall 1996) occurred in thesame province. GP and L genes of EBO virus isolates from all three epidemics were partiallysequenced, which showed a difference of 0.1% in the base pairs. Sequencing also showed that allisolates were very similar to subtype Zaire EBO virus isolates from the Democratic Republic of the

Congo.

Johnson et al. [1] isolated and identied Ebola (EBO) virus associated with a mortality rate similar to that seen during theDRC and Sudan epidemics [7].from human cases during a 1976 epidemic of hemorrhagic

fever (HF) in the Democratic Republic of the Congo (DRC). During 1994 and 1995, an outbreak of HF occurred in north-eastern Gabon. It was rst considered to be caused only byDuring the same year, EBO virus was isolated from patients

during an HF epidemic in Sudan [2]. The viruses, which were yellow fever (YF) virus on the basis of the clinical symptom-atology, routine biochemical tests, and specic laboratory re-closely related to Marburg virus (all members of the Filoviri-

dae), had an 88% and a 53% case fatality rate in DRC [3] and sults provided by the Centre National de Re ference des Fie vresHemorragiques Virales (InstitutPasteur, Paris) [8, 9]. However,Sudan [2], respectively. A third outbreak, with a case fatality

of 60%, occurred in Sudan in 1979 [4]. In addition, a death retrospective serologic tests detected concomitant EBO virusantibodies among some of the patients and the general popula-was registered in Tandala, DRC, in 1979 [5]. In 1994, a new

strain of EBO virus was isolated from a Swiss researcher with tion [10, 11]. Later efforts to isolate the EBO virus from someof the specimens from the rst epidemic (1994) were success-a dengue-like syndrome, who had likely been infected duringthe necropsy of a chimpanzee (see Formenty et al., this supple- ful. One year later, in February and in July 1996, two more

HF outbreaks occurred in northeastern Gabon [1013].ment). The animal had been found dead in the Ta FNationalForest (Co te dIvoire) during a 2-year epidemic that killed half Herein, we report on three EBO epidemics that occurred

between late 1994 and early 1997 in northeastern Gabon [10].of the population of chimpanzees [6]. A third serious humanepidemic of EBO occurred in 1995 in Kikwit, DRC; it was

The Epidemics

It is important to note that during this investigation, weInformed consent was obtained from the patients or their parents or guard-continually faced many difculties (e.g., logistics problems and ians.

Financial support:CIRMF is supported by the Republic of Gabon, the French cultural and political constraints) in the collection of data and Ministry of Foreign Affairs (Coope ration et Action Culturelle), and ELF GA-

management of our research on this disease. Despite our efforts,BON Co. Ltd. (Libreville). the difculties sometimes led to the loss of important informa-Reprints or correspondence (current afliation): Dr. Alain-Jean Georges,Chefferie, Hopital dInstruction des Arme es Desgenettes, 108 Bd Pinel, 69 tion, and at times, forced us to report the scientic data in a275 Lyon, Cedex 03, France ([email protected]). rather unorthodox manner.* Current afliations: CHA, Lamalou les Bains, France (J.P.M.L.); IMTSSA,

First epidemic (fall 1994). The rst epidemic in GabonLe Pharo, Marseille Arme es, France (E.J.B.); Ministe `re Affaires Etrange`res,Service de lAction Humanitaire, Paris (J.P.A.); and CBMS, Institut Pasteur, had two waves of patients, with the rst beginning in earlyParis (M-C.G.C.). December 1994 and the second beginning at the end of JanuaryThe Journal of Infectious Diseases 1999;179(Suppl 1):S6575 to February 1995. All patients in the rst wave came from 31999 by the Infectious Diseases Society of America. All rights reserved.00221899/99/79S10013$02.00 gold-panning encampments (Me kouka, Andock, and Minke be)

/ 9d49$$se13 01-12-99 11:06:39 jinfa UC: J Infect


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S66 Georges et al. JID 1999;179 (Suppl 1)

situated in small clearings of 20003000 m 2 at the edge of the the physicians in charge of the Makokou hospital (a very poorlyequipped facility), was rather broad, and at least 4 of the 49rain forest. Figure 1 and table 1 show the geographic location

of these and other villages with case-patients. Table 1 also patients appeared unlikely to have HF. On 18 December 1994,during the middle of the rst wave, the Gabonese health author-shows the chronology of illness for the cases. Three hundred

fty people, mainly of the Bakota ethnic group but also some ities requested that we examine 9 patients in Makokou GeneralHospital. Biologic samples from those patients led to the identi-Bakwe le, inhabit this area (30 in Me kouka, 20 in Andock, and

300 in Minke be). cation of the etiologic agents.

Between December 1994 and March 1995, CIRMF collected A total of 32 sick persons from the three forest encampments(23 from Me kouka, 4 from Andock, and 5 from Minke be) 22 samples in Mayela from contacts (all 15 years old) of

sick persons and 88 samples from the local population of traveled 100 km south by river to the nearest hospital at Mako-kou, the main town of the region, for treatment. In addition to Ogooue -Ivindo Province, Gabon, where the epidemic occurred.

At that time, many people had ed the area in terror; therefore,the primary cases in Andock, we were also informed that deathshad occurred in the local population of great apes (chimpan- it was difcult to conduct an investigation. A year later (January

1996), we sampled 236 people from the three initially infected zees, Pan p. troglodytes , and gorillas, Gorilla g. gorilla). How-ever, despite intensive searches of the forest by teams from villages and from villages in the Makokou area in order to

further assess exposure to EBO virus in the local population.Centre International de Recherches Me dicales de Franceville(CIRMF), no cadavers or skeletons were ever found, even when Second epidemic (spring 1996). A second epidemic began

during early February 1996 in the village of Mayibout 2, Ga-a patient told us that he had killed a chimpanzee with abnormal behavior inside his encampment. This report could be anec- bon, which is located on the Ivindo River. Mayibout 2 is 40

km south (6 h by boat) of Me kouka and Andock, where thedotal, but it cannot be totally ignored.The second wave of patients did not originate from the en- rst epidemic broke out, and north of Makokou (7 h by boat).

Eighteen people who had skinned and chopped a chimpanzeecampments: They were what we should have called (in a surveyfollowing accepted procedures) secondary (or tertiary ?) cases. cadaver that they found became ill (fever, headache, bloody

diarrhea). They were evacuated from Mayibout 2 to MakokouUnfortunately, the information that was available from the au-thorities regarding these patients did not provide a precise gene- on the decision of the village chief, despite governmental in-

structions to the contrary. All patients were admitted to Mako-alogy of all cases. The rst patient in the second wave wasfrom Mayela, a small village close to Makokou, far from the kou Ge neral Hospital, where 4 moribund patients died within

48 h.forest encampments. This person, who was probably the rstsecondary or tertiary case, lived near a traditional healer (a The bodies of the 4 patients were returned by river to Mayi-

bout 2; a fth patient, who was moribund when he escaped nganga) and was probably infected as a result of contactwith a hospitalized patient who, against medical advice, left from the hospital, died after returning to Mayibout 2. Tradi-

tional burial ceremonies were performed without any specialthe hospital to seek care from the nganga. Sixteen more cases(table 1) occurred in mid January: 1 case at Makokou Ge neral precautions to avoid disease transmission. Two other primary

cases occurred, which appeared not to be connected to theHospital, 12 cases from Mayela, and 3 cases from two villages(Ekataniabe , 1 patient; Ekobakoba, 2 patients) on the road chimpanzee episode; 1 died. Fifteen serum samples from these

initial 18 primary cases and 6 from secondary and tertiary casesrunning south toward Franceville. None of these patients had been in the area affected by the rst wave of the epidemic were obtained. An additional 205 serum samples were obtained

from the population of Mayibout 2 and neighboring villagesduring the previous 3 months; however, all had been either indirect contact with sick relatives (people hospitalized at Mako- (Mayibout 1 and Mvadi, which are 2 km south and 20 km

north, respectively, of Mayibout 2).kou General Hospital or sleeping in the nganga traditional heal-ers home) or with people caring for patients. Third epidemic (fall 1996). On 5 October 1996, we in-

formed the Gabonese health authorities (who 1 week earlier The last reported case (infected while caring for a relativeat Makokous hospital) occurred at Ekobakoba on 9 February had requested our assistance in the investigation) that we had

isolated EBO viruses from 2 of 6 samples from patients hospi-1995, and the epidemic was declared over by Gabonese healthauthorities on 17 February 1995. talized at Booue . Personnel from CIRMF carried out a retro-

spective investigation of this third epidemic, which probablyOverall, 49 persons were admitted to Makokous hospitalwith suspected HF. Patients were suspected of having HF if started as early as 13 July with the death (undeclared) of a 39-year-old hunter in a logging camp near Booue (0 06 S, 11 57they had a well-identied contact with an ill person or at least

two of the following clinical signs: fever, diarrhea, vomiting, E) between Ovan and Koumameyong, 200 km from Me koukaand 120 km from Makokou to the southwest (gure 1). Themelena, conjunctivitis, arthralgia or myalgia, diarrhea, or vom-

iting. Of the rst 9 patients, 2 who we examined during the symptoms of this rst case were suggestive of viral HF (VHF;fever, bleeding, vomiting, diarrhea, and headache). At the be-rst wave also had jaundice, which has never been described

as a symptom of EBO infection but is consistent with YF ginning of August 1996, information was obtained that severalchimpanzees may have died in the same area, and a eld collab-infection. This case denition of HF, which was proposed by



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Figure 1. Geographic location of sites of primary and secondary cases of hemorrhagic fever. A , Gabon, B, Africa, C , sites.



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Table 1. Geographic location (in order of occurrence of illness) of sites of primary and secondaryhuman Ebola infections during the fall 1994 outbreak in Gabon.

Samples from Samples from Samples fromsick patients patients contacts general population

Village Geographic location (rst epidemic) (rst epidemic) (1996)

Mekouka 1 24 N 12 59 E 23 12

Andock 1 29 N 12 55 E 4 8Minke be 1 44 N 12 49 E 5 86Mayela (December 1994) 0 38 N 12 56 E 22 12Makokou 0 33 N 12 50 E 1 28 72Mayela (February 1995) 0 38 N 12 56 E 13 32 24Etakaniabe 0 32 N 12 57 E 1 12 12Ekobakoba 0 36 N 13 05 E 2 1 10Mayibout 2 1 07 N 13 06 E 175Mayibout 1 1 07 N 13 06 E 10Mvadi 1 46 N 13 09 E 20

orator was able to obtain skin samples from 1 cadaver for The epidemic was declared over in March 1997 (last of-cially declared patient: 18 January), with a total of 60 cases pathologic examination at the Centers for Disease Control and

Prevention (CDC, Atlanta), with which CIRMF was in contact. and 45 deaths. Unfortunately, the information made availableto us from the local authorities did not allow for a very preciseSix weeks later, at the end of August, a second hunter (35

years old) died in the same Gabonese logging camp, but his genealogy of all cases; however, we did obtain accurate dataon 47 of the 60 cases (Milleliri JM, unpublished data).death, which was also suggestive of VHF, was not linked to

that of the rst hunter. A third hunter (26 years old) becameill 12 days later and was hospitalized at Booue . He subsequently

Materials and Methodsevaded the medical authorities at Booue and died in the villageof Balimba, where he was being treated by a nganga. Shortly Routine Clinical Chemistry Testsafter the hunters death, both the nganga and his nephew fell

Routine clinical chemistry tests were done on sera from someill in mid-September and were admitted rst to Booue Hospitalhospitalized cases in each epidemic.and then transferred to Makokou General Hospital, where they

died. Three other cases, including another nganga, occurred among people living in the same area.

Serologic AssaysAfter we conrmed that the patients had EBO HF (EHF), IFAs. IFAs were used to detect viruses causing HF (includingthe authorities decided to apply the same clinical and epidemio-

Crimean-Congo HF fever virus, Rift Valley fever virus, subtypelogic denitions that were used during the outbreaks in Yam-Zaire of EBO virus [EBO-Z; Mayinga and Boniface strains], Lassa buku, DRC, in 1976 and in Kikwit to diagnose EHF patientsfever virus, and Marburg virus) in sera from some patients in each

[1, 7]. The disease spread around Booue ; 24 cases, including epidemic. The IFAs were done using CRELM (initials of each17 deaths, were ofcially declared by 13 November 1996. A virus) microscope slides [14] provided by the CDC and US ArmyGabonese doctor, who had performed an endoscopy in a private Medical Research Institute of Infectious Diseases (Fort Detrick,hospital in Libreville on an EBO-infected patient from Booue , Frederick, MD).developed signs and symptoms of EHF. On 27 October, without IgM anti-EBO EIA. ELISAs were also used to detect EBO

virus infection [15]. For IgM detection, an immunocapture testrst requesting a serologic diagnosis of VHF and without con-was used. Polyvinyl chloride 96-well microtiter plates (no. 1000-3,sidering the possible etiology of such a diagnosis, he went toBioblok; Dynatech, Strasbourg, France) were adsorbed overnightJohannesburg for treatment. A South African nurse who cared at 4 C, using goat anti-human m-chain (Tago, Burlingame, CA)

for the doctor became ill on 2 November and died on 24 diluted 1:500 in 0.01 M PBS (pH 7.4). All steps of the assay were November (Swanepoel R, personal communication).done using volumes of 100 m L/well. Sera from persons who wereAt the end of November 1996, a second wave of this third infected or were suspected of being infected and sera from their

epidemic appeared in three locations around Booue : Lolo (3 contacts were added to the wells at a 1:100 dilution in serdil (5%deaths, 6 cases), SHM, a timber company (4 deaths, 5 cases), skim milk [Bacto milk], PBS, 0.1% Tween 20). After the platesand the logging camp of Balimba (1 death, 1 case). A conrmed were washed with PBS with 0.1% Tween 20 (Bio-Rad, Richmond,case spread to Lastourville (130 km southeast of Booue ) from CA), sera were added (rst at a 1:100 dilution and then at 4- toBalimba, and again to Libreville, with subsequent transmission 800-fold dilutions), and the plates were incubated for 60 min at

37 C in a humid chamber. For each serum and each dilution, inof the disease to the capital (11 deaths, 15 cases).



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S69JID 1999; 179 (Suppl 1) Ebola Outbreaks in Gabon, 1994 1997

one well, we used as antigen a cell slurry made in EBO-infected known from previous experience to identify EBO antigen for dif-ferent strains of virus in tissues. Molecular identication of theVero E6 cells diluted 1:1000 in PBS; in another well, we used a

slurry of uninfected Vero E6 cells. Anti-EBO (subtypes Z, Sudan EBO virus strains following extraction of RNA from whole blood clots or from the peripheral blood mononuclear cells separated on[S], and Reston [R]) hyperimmune rabbit serum was diluted 1:2000

in serdil. Plates were incubated for 1 h with anti-rabbit IgG (no. coll have been described elsewhere [16].At Institut Pasteur, attempts to isolate YF virus by inoculation074-1506; Kirkegaard & Perry, Gaithersburg, MD) diluted

1:10,000 in serdil (as conjugate) and then incubated for 30 min of suckling mice, mosquitoes, and mosquito cell cultures (AP 61,C636) failed to give positive results. Therefore, RNA was ex-using an H 2 O2 0 ABTS substrate (Kirkegaard & Perry).

In the rst assays we used 2 standard serum samples as positive tracted according to the method of Chomczynski and Sacchi [18],and the presence of the YF viral genomic RNA was revealed bycontrols. As soon as we had conrmed the EBO cases, we added

our own 2 positive controls, which were run in standard dilution reverse transcriptase polymerase chain reaction (RT-PCR) ampli-cation [19] using consensus primers for aviviruses. The ampli-series for providing standard curves, which allowed the determina-

tion of the limits of detection of the assay. A panel of 4 negative ed DNA fragment was then transferred onto a nitrocellulosemembrane and hybridized with a YF virusspecic probe.sera (from healthy persons) were run in all tests to determine the

background of the assay (the limit at which the positive controlsare positive) and to provide the mean and standard deviation (SD)

Antigenemiaof that background. A sample was considered to be positive if itsoptical density exceeded the mean / 3 SD of the controls. Plates

The sera of sick people, their contacts, and people suspected of were read spectrophotometrically at an optical density of 410 nmhaving EHF were tested by EIA for the presence of EBO antigen,on a microcomputer (LP 200; Sano Diagnostics Pasteur, Marnes-as were the supernatants of Vero E6 cells seeded with sera fromla-Coquette, France).these persons. Half of a 96-well microtitration plate was coated IgG anti-EBO EIA. For IgG detection, half of a 96-well mi-(overnight at 4 C) with a mouse monoclonal antibody recognizingcrotitration plate was coated with a lysate of EBO-Z, EBO-S,EBO virus epitopes; the other half was coated with normal my-and EBO-Rinfected Vero E6 cells, and the other half was coated eloma ascites diluted 1:1000 in PBS. Biologic samples to bewith a lysate of uninfected Vero E6 cells to determine the specicchecked for the presence of EBO antigen were then added (rst binding of antibody to virus antigens (the background); both ly-at a dilution of 1:4 and then 4- to 64-fold). After a 1-h incubationsates were diluted 1:1000 in PBS. Plates were incubated overnightat 37 C in a humid chamber, the plates were washed 3 times, and at 4 C. The plates were washed three times with PBS plus 0.1%rabbit anti-EBO (subtypes Z, S, and R) diluted 1:2000 in serdil(vol/vol) Tween 20, and then sera were added at a 1:100 and 4-was added and incubated for 1 h at 37 C in a humid chamber.to 800-fold dilutions in serdil and allowed to react with the antigen-Plates were incubated for 1 h with anti-rabbit IgG conjugate (no.coated wells for 60 min at 37 C in a humid chamber. Bound IgG074-1506; Kirkegaard & Perry) diluted 1:10,000 in serdil and thenwas detected with mouse anti-human IgG conjugated to horserad-incubated for 30 min using an H 2 O2 0 ABTS substrate (Kirke-ish peroxidase. Further identication was done as in the IgM EIAgaard & Perry), and bound rabbit IgG was detected.assay. A panel of specic IgG-positive controls and specic 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

Resultsof the adjusted optical density were accumulated and used to calcu-late a value equal to the mean / 3 SD, which represented the Serologic surveys. The occurrence of HF cases in the fallcutoff value. Plates were read as above. of 1994 initially led us to suspect an outbreak of sylvatic YF

Anti-YF IgM EIA. At the beginning of the rst VHF epidemic, infection. However, we were aware that another HF agent (inYF virus was strongly thought to be the probable cause of illness, particular EBO virus) could be circulating, given the possibleon the basis of the clinical and epidemiologic results of the 9

fatalities observed in nonhuman primates (apes replicate YF patients examined. Therefore, we tested for YF virus infection,virus, but they usually do not die from it; Monath TP, personalusing IgM capture techniques [17].communication).

In the rst instance, we screened 9 patients (all from Mako-kou General Hospital) for HF virus. EBO IFA on CRELMVirus Isolation Attemptsslides was negative for all 9 serum samples, whereas 6 of 9

Vero E6 cell 6-well plates were inoculated with sera from pa- patients showed evidence of recent YF virus infection (IgMtients suspected of harboring a VHF virus. During the rst epi-

capture). Of the 49 sick patients, another 14 cases who had demic, the biosafety level 4 facilities of the Institut Pasteur were never been vaccinated against YF were also screened. Of theused for all virologic manipulations of patient-derived material; 14 cases, 8 had evidence of recent YF infection as determined further work on the two last epidemics was carried out at CIRMFs

by IgM capture or Southern blot, but none had evidence of site or at CDCs Special Pathogens laboratories or at both sitesantibodies to the main African viral HFs as determined by usefor part of the molecular biology testing of EBO virus strains [16].of IFA on CRELM slides. However, the antigen used in our To identify the EBO virus, EBO reference antisera were tested IFA test was several years old, so in June and July 1995 weagainst the virus isolates cultured on Vero E6 cells. Tests wereused ELISAs and fresh antigens to retrospectively test thesedone using polyclonal antibodies raised in mice (IFA test) and a

pool of monoclonal antibodies (antigen-capture test) that were same 23 samples (ELISA is generally more sensitive than IFA).



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Nineteen patients had signs of a recent infection with EBO as For the spring 1996 epidemic, the case fatality rate was67.7% (21 of 31 cases died) as determined using the accepted determined on the basis of at least one of the following: positive

for EBO IgM, positive for antigenemia, positive for virus iso- (Yambuku and Kikwit) case denition. There were 17 maleand 14 female patients (sex ratio, 1.2) with a mean age of 27.6lated from cultured Vero E6 cells.

We also used ELISA to test the 88 serum samples obtained years.In the fall 1996 epidemic, there were 60 cases and 45 deathsin January 1995 (before YF vaccination) from the population

living around the three encampments (Andock, Minke be, and (case fatality rate, 75%). The sex ratio was 2.4. We were unable

to conduct adequate surveys during the epidemic, so completeMekouka) where the rst epidemic broke out and the 22 serumsamples obtained from contacts in Mayela. Two of these sera age-range data are unavailable.

The development (geographically and chronologically) of the(1.8%) were positive for anti-EBO IgG but not EBO IgM,while 9 YF-unvaccinated people (8.1%) were positive by YF rst two epidemics is shown in table 1 and gures 2 and 3,

respectively.When tracing the computerized (MSAccess software;IgM capture, indicating a recent YF virus circulation.One year later, we obtained samples from 236 persons from Microsoft, Redmond, WA) polynomial regression curves obtained

from histograms, the rst epidemic shows two waves of morbidity,the same area (table 1). For various reasons, only 56 of theoriginal 110 people were part of this second cohort (e.g., use whereas in the second outbreak, there is only one peak of morbid-

ity. These observations are related to the notably different typesof given name in the place of family name in original survey,fear of being identied and accused of causing the original of spread in the two epidemics. Persons in several villages were

infected during the rst epidemic, which lasted longer than theepidemic, and migrations of populations). Of 236 subjects, 24(9.7%) had anti-EBO IgG by ELISA without any anti-EBO second, in which only persons in a conned area were affected.

The exact origin(s) of the virus in the rst epidemic is uncertain,IgM (1 of these IgG-positive cases was the rst case of therst wave in the 1994 epidemic and had survived a typical and despite reports of the death of great apes, we were unable to

conrm these reports.EBO-type illness).During the second epidemic, 205 samples were collected Exploitation of the area by gold miners has caused substan-

tial disturbance to the forest canopy. This disturbance mayfrom people living in 3 villages (Mayibout 1 and 2, whereEBO was known to have occurred, and Mvadi, where 1 uncon- have caused contact between other species and humans that

otherwise would not have occurred. None of the specimensrmed death of a hunter was said to have occurred). Highfrequencies of anti-EBO IgG (14.9%30%) and IgM (5.7% (small mammals and insects) collected in the eld so far have

yielded any sign of harboring the virus; however, these studies10.0%) by ELISA in all three villages (table 2) may suggestthe presence of an asymptomatic or mild form of the disease are ongoing.

In the second outbreak, there is no doubt that the virus cameconcomitant with the classical severe form of EBO, or theymay demonstrate the limits, in terms of specicity, of the EBO from one source, a chimpanzee infected by EBO (for which

the chain of infection is unknown). The chimpanzee seems toIgG and IgM ELISAs that we used.During the initial phases of the third epidemic, we were have been the index case for infecting 18 primary human cases.

There were 2 other primary cases, for whom we have no infor-unable to carry out serologic surveys due to local reasons thatwere out of our control. mation on the source of infection (the role of a nganga was

suspected), making a total of 20 primary cases for this epi- Fatality and descriptive epidemiology. We reviewed lesat Makokou General Hospital for persons who were seen during demic. Only those people who had been in contact with the

dead chimpanzee before it was cooked for eating were affected;the rst epidemic in fall 1994; 49 patients were identied withat least two signs of VHF, 29 of whom died (case fatality rate, nobody was infected by eating the cooked meat. We identied

10 secondary cases involving close contacts of dead or dying59%). There were 26 male and 23 female patients (sex ratio,1.1) with a mean age of 37 years. 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 thesecond day of the epidemic. All equipment and material neces-Table 2. Anti-Ebola antibodies among the general population of sary for barrier nursing and prevention of the spread of diseasethree villages in Ogooue -Ivindo Province, Gabon, during the second

epidemic (spring 1996), as determined by ELISA. were provided. One hundred ninety-one contacts were traced,with no evidence of infection by EBO virus. No. (%) of villages Similar to the case in the rst epidemic, the source(s) of the

positive for virus in the third epidemic was not immediately obvious, nor Geographic No. of could it be traced retrospectively. The lack of identication of Village location samples IgG IgMcases at the outset and the lack of adequate containment mea-

Mayibout 1 1 07 N 13 06 E 10 3 (30.0) 1 (10.0) sures once the diagnosis had been made meant that sick personsMayibout 2 1 07 N 13 06 E 175 26 (14.9) 10 (5.7) could travel throughout and even outside Gabon. Some contactMvadi 1 46 N 13 09 E 20 5 (22.5) 2 (10.0)

cases could not be identied and isolated.



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Figure 2. Evolution of 19941995 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.

Immunostaining [15] done at the CDC on skin biopsies from tients in the cohort, 6 died. Data were obtained 4 7 days after the onset of disease (16 days after the patients were infected a chimpanzee found dead in the forest indicated the presence

of EBO [16]; however, the ape is very unlikely to have had by a chimpanzee cadaver). The levels of direct and conjugated bilirubin were normal, but all patients had signs of liver any contact with humans.

Biologic and clinical features. Clinical signs and symp- involvement (i.e., elevations in g -glutamyltransferase, amino-transferase, and alkaline phosphatase), and 23% had signs of toms observed in 15 serologically or virologically conrmed

patients from the outbreak at Mayibout 2 (spring 1996) are slightly decreased renal dysfunction (elevated serum creatinineand urea). presented in table 3. Fever was always present, while in 75%

of the cases, diarrhea (a dense suspension of black or brown- Most of the other blood parameters (e.g., total serum pro-teins, albumin, uric acid, cholesterol, triglycerides, sodium, po-red uneven microaggregates 5 mm in size in liquid or syrupy

stools) and vomiting were seen, and 50% had conjunctivitis tassium, and chloride) that we were able to test were normal.Characterization of virus strains. EBO viruses were iso-or conjunctival bleeding. Taking only the second epidemic

into consideration, since it was the best-documented outbreak, lated from patients from each of the three epidemics. All theGabonese virus strains sequenced showed a high level of ho-clinical signs and symptoms were observed between days 6and 11 after handling the chimpanzee, and deaths occurred mology with the group of strains from DRC [16]. Viruses

isolated from patients during the same outbreak had identical between days 12 and 18 after the appearance of signs or symp-toms. Despite a small number of observations, it seemed to us base-pair sequences, but base-pair sequences were slightly dif-

ferent for isolates from the individual outbreaks. There wasthat those who died had a shorter incubation period than thosewho survived. 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 Table 4 presents the biologic and biochemical data for 13 patients infected during the spring 1996 outbreak. Of 13 pa- 1994 strain differs from that of the February 1996 strain by



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Figure 3. Evolution of spring 1996 Ebola virus epidemic in Gabon. A, Evolution of epidemic by mortality rate. Curve shows monophasicevolution 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.

four nt, and the February strain differs from the October 1996 Discussionstrain by 4 nt).

An epidemic of HF among humans, some of whom had A conserved region of the L gene was also amplied in virusclinical and biologic symptoms suggestive of YF infection,strains from fall 1994 and fall 1996, using degenerate primersstarted in December 1994 in Ogooue -Ivindo Province. On theselected from sequences of EBO-S and Marburg virus strains; basis of clinical, biologic, and serologic data, a diagnosis of PCR products were subsequently sequenced [13]. The 2 Gabo-YF infection was suspected in 6 hospitalized cases, and a diag-nese strains showed high homology in the rst 156-bp se-nosis of YF infection was conrmed for 3 other cases on thequence, with only a single base change (position 110; A to G) basis of molecular biology data, although the complete virusseparating the 2 strains. As stated, during the rst epidemic, 6

could not be isolated, which is not unusual for YF virus. (YFof 9 patients had anti-YF IgM. At the Institut Pasteur, using virus is known to be impossible or difcult to isolate fromPCR and specic hybridization techniques, YF sequences wereclinical material if samples are taken several days after evolu-identied in sera from 2 of the 6 patients who were anti-YFtion of the clinical disease; for example, during the 1970 epi-IgM positive and in 1 of the 3 patients who had no anti-demic in Okwooga and the 1986 epidemic in Oju region, Nige-YF IgM. Those 3 patients, who were negative for all EBOria, it was impossible and difcult, respectively, to isolate YFinvestigations, had clinical signs compatible with YF (e.g.,virus [Monath TP, personal communication]). At the time, nosevere low back pain, fever, jaundice [2/3 patients], melena,

and melanemesis), and biochemical results showed signs of etiologic diagnosis was scientically established for the 6 pa-tients who were not conrmed to have YF virus infection;liver and renal failure and slightly raised bilirubin levels.



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Table 3. Clinical manifestations observed in 15 conrmed cases of of infected case-patients, with barrier nursing, disinfection,Ebola hemorrhagic fever during the spring 1996 outbreak in Mayibout and insect eradication programs and (2) active reintroduction1 and Mayibout 2, Gabon. of a vaccination program against YF virus, which had lapsed

10 years earlier. The fact that the entire YF virus was notClinical symptoms % of patients with symptomisolated is not a conclusive argument against the fact that YF

Fever 100 occurred during the rst VHF epidemic, since samples mayDiarrhea 87 have been collected at a late stage of the disease when theVomiting 73 virus is difcult to isolate. However, in fall 1994, there was aConjunctivitis 53

high prevalence of anti-YF IgM in YF-unvaccinated subjects,Gingival bleeding 40which was strongly suggestive of a recent YF outbreak. It isArthralgia, myalgia 33

Cephalgia 27 known that sylvatic YF infection can exist in a mild or symp-Asthenia 27 tom-free form [17].Cephalgia 27 A nal argument for the presence of YF virus during theMelena 27

rst epidemic (fall 1994) comes from Pisano et al. [20], whoHiccups 27identied YF infection in a cohort of 5 patients (different fromObnubilation 27

Sore throat 20 ours) from the same area by sequencing a cDNA fragmentCough 20 obtained by RT-PCR and showing 94.4% homology with theMelanemesis 13 Asibi strain of YF virus [21].Epistaxis 13

During the rst epidemic, EBO and YF viruses were obvi-Abdominal pain 7ously 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

nevertheless, we felt, from a public health point of view, that virus alone or in combination, but a nosocomial amplicationthere was a risk for reemergence of YF (the last epidemic in of EBO appears to have occurred inside Makokou Ge neralGabon was in 1949 [8]) among the unvaccinated population, Hospital. Such an association of YF and EBO viruses duringas conrmed later by the epidemiologic investigations of a the same epidemic has been suspected in the past: In Ethiopia,World Health Organization expert [9]. where a VHF outbreak occurred in 19611962, a later sero-

Without waiting for a denitive diagnosis, for which inves- logic retrospective study showed that both EBO and YF virusestigations were in progress at the hemorrhagic fever unit at may have been present [22].the Institut Pasteur, and despite the fact that another etiologic Earlier IFA-based studies looking for the presence of EBOagent could be involved, CIRMF suggested to the Gabonese virus in other provinces of Gabon were done by Ivanoff et al.health authorities that they rapidly instigate measures to limit [23] in 1982 and Meunier et al. in 1986 [24]. Data obtained

by use of the same technique showed that EBO is widespread the spread of YF infection. We suggested (1) passive isolation

Table 4. Biochemical results for 13 patients with Ebola hemorrhagic fever during the spring 1996 outbreak in Gabon.

Conjugated Aspartate Alanine AlkalinePatient No. of days Total bilirubin bilirubin g -glutyltransferase aminotransferase aminotransferase phosphatase Ureano. after onset Outcome ( 20 mmol/L) ( 10 mmol/L) ( 45 U/L) ( 45 U/L) ( 45 U/L) ( 125 U/L) (2.5 7 mmol)

1 7 R 52.0 35.0 246.0 230.0 666.0 419.0 11.02 4 D 42.0 30.0 212.0 220.0 512.0 410.0 10.93 4 R 17.0 4.7 16.0 155.0 60.0 126.0 3.14 4 D 25.0 6.7 52.0 160.0 75.0 166.0 3.15 6 D 16.5 4.4 64.0 314.0 109.0 150.0 3.26 4 R 18.0 7.2 38.0 226.0 98.0 72.0 6.77 4 D 21.0 6.7 21.0 165.0 70.0 136.0 3.28 4 R 31.7 19.8 193.0 185.0 1499.0 637.0 17.99 5 R 17.9 7.0 30.0 219.0 88.0 69.0 6.5

10 4 D 14.8 3.5 21.0 135.0 74.0 59.0 2.711 4 R 15.0 3.7 26.0 140.0 76.0 72.0 5.212 4 R 14.2 9.9 15.0 266.0 50.0 82.0 1.313 4 D 19.8 9.0 23.0 56.0 22.0 66.0 3.1

Mean 23.5 11.4 73.6 190.1 261.5 189.5 6.0SD 11.7 10.3 83.7 65.9 420.5 181.8 4.7

NOTE. Data in parentheses are normal values. R recovered, D died.



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throughout equatorial Africa [25 27]. However, these results to limit the spread of this disease. Nosocomial spread of infectiondue to the lack of recognition of the disease and the dearth of must be examined carefully since others have suggested that

IFA is likely to give false-positive results [28]. In addition, in training and material available to establish simple barrier nursingremains the root cause of most of the disease observed to date.our hands, this test was insensitive for EBO virus, specically

during the fall 1994 epidemic, in which all sera collected during From the public health point of view, this remains a basic goal for the prevention of EHF epidemics. Preventing primary infectionthe acute phase of the disease tested negative by IFA, while

some were positive by EIA. Even though ELISA techniques certainly requires the identication of risk factors and the reser-

voir, something that continues to elude us all.appear to be very sensitive for assessing EBO infection, wemust be cautious about their specicity.

No serologic data were available from persons in Ogooue -AcknowledgmentsIvindo Province before the rst epidemic. However, we had

kept 58 untested frozen serum samples from the epidemic pe- We thank B. Le Guenno (Institut Pasteur, Paris) for his invalu-riod. The samples were from asymptomatic adults who were able scientic assistance in afrming the yellow fever diagnosisnot associated with gold-panning; 2 of them had ELISA EBO in January 1995 and for initiating new retrospective serologic stud-IgM, suggesting recent EBO infection, and the remaining 56 ies on Ebola virus in June 1995 and new attempts at virus isolation

in samples from the fall 1994 epidemic, which led to the isolationsamples were negative for anti-EBO IgG and IgM. This lowof the rst Ebola isolate from Gabon; P. Tshipamba (CIRMF) for percentage of seropositivity suggests that the circulation of providing excellent technical assistance; J. B. McCormick (InstitutEBO is a new phenomenon in the area, affecting mainly peoplePasteur), S. Fisher-Hoch McCormick (Fondation Me rieux, Lyon,living deep in the forest in temporary encampments.France), and P. Rollin (CDC, Atlanta) for valuable and pertinentThere are interesting demographic and ecologic differencessuggestions regarding the manuscript; and members of the Special between the sites of the rst two epidemics. The gold panners Pathogens Branch of the CDC as well as members of Walter Reid

in the fall 1994 outbreak were a transient population and caused Army Institute of Research, US Army Medical Research Instituteconsiderable damage to the local forest in which they were of Infectious Diseases (Fort Detrick, Frederick, MD), who haveworking. Most of those who survived left the area after the generously provided reagents to CIRMF since 1982.epidemic; a very small population remains. Mayibout 2, a per-manent, relatively dense settlement whose inhabitants practice

Referencesslash-and-burn cultivation, is situated on the river Ivindo and is surrounded by forest. The disturbance of the forest integrity 1. Johnson K, Webb P, Lange J, Murphy F. Isolation and partial characterisa-

tion of a new virus causing acute haemorrhagic fever in Zaire. Lancetis one common feature between the two sites, but the one1977 ;1:56971.important difference is the permanence and the size of the

2. WHO/International Study Team. Ebola hemorrhagic fever in Sudan, 1976. population around Mayibout 2, which might permit the virus,Bull World Health Organ 1978 ;56:27193.

once present in the area, to circulate within the human popula- 3. WHO/International Study Team. Ebola hemorrhagic fever in Zaire, 1976.tion. Both the reservoir and the vector for the EBO virus are

Bull World Health Organ 1978 ;56:24770.4. Baron RC, McCormick JB, Zubeir OA. Ebola virus disease in southernstill unknown; however, it is apparent that the great apes, or Sudan: hospital dissemination in intrafamilial spread. Bull World Healthat least chimpanzees, are not involved because they are asOrgan 1983 ;61:9971003.susceptible as humans to the disease.

5. Heymann D, Weisfeld, Webb P, Johnson K, Cairns T, Berquist H. EbolaOn the basis of a sizeable but incomplete genetic compari- hemorrhagic fever: Tandala, Zaire, 19771978. J Infect Dis 1980 ;142:

son, the viruses from our three outbreaks are essentially indis- 3726.6. Le Guenno B, Formenty P, Wyers M, Gounon P, Walker F, Boesch C.tinguishable from the viruses isolated in DRC in 1976 and

Isolation and partial characterisation of a new strain of Ebola virus.1995; however, the viruses isolated in 1976 from Sudan and Lancet 1995 ;345:12714.DRC were clearly biologically distinct from one another [29].

7. WHO/International Study Team. Ebola hemorrhagic fever in Zaire (Kik-We cannot rule out the possibility that varied strains of EBO wit). Bull World Health Organ 1995 ;19:13.virus or even different members of the Filoviridae are circulat- 8. World Health Organization. Yellow fever in Gabon. Bull World Health

Organ 1995 :64.ing in these areas, with some being more virulent than others9. Cordelier R. Fie`vre jaune au Gabon: enquete sur un e pisode amaril dansyet cross-reacting and, therefore, appearing as asymptomatic

la Province dOgooue-Ivondo (2 au 23 mars 1995). Rapport OMS.infections in the population. The inability to isolate viruses Brazzaville: OMS, Bureau Re gional pour lAfrique, 1995 .from the Gabonese environment and from anyone but ill pa- 10. Georges AJ, Le Guenno B, Renaut AA, et al. Recent Ebola virus outbreakstients has not allowed us to address this issue. The fact that in Gabon from 1994 to 1996: epidemiologic and control issues. Report

of International Colloquium on Ebola virus research (Antwerp, Bel-the ELISA test has not been evaluated for specicity againstgium). Antwerp: Institute of Tropical Medecine, 1996 :47.known human infections also does not permit us to eliminate

11. Amblard J, Obiang P, Edzang S, Prehaud C, Bouloy M, Le Guenno B.this as a possible explanation for the high level of antibody inIdentication of the Ebola virus in Gabon in 1994. Lancet 1997 ;1:

the population of Mayibout 2. 1812.Finally, it is very clear that the use of simple barrier nursing 12. World Health Organization. Ebola hemorrhagic fever in Gabon. Bull

World Health Organ, 1996 ;9:71.methods in even the most basic of hospital settings is sufcient



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j Infect Dis.-1999-Georges-s65-75 (Article Ebola Virus)

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