THE LANCET

Galai N, Vlahov D, Margolick JB, Chen K, Graham NMH, Muoz A.

Changes in markers of disease progression in HIV-1 seroconverters: a
comparison between cohorts of injection drug users and homosexual
men. J Acquir Immun Defic Syndr 1995; 8: 6674.
Pezzotti P, Phillips AN, Dorucci M, et al, and the Italian
Seroconversion Study Group. Category of exposure to HIV and age in
the progression to AIDS: longitudinal study of 1199 people with
known dates of seroconversion. BMJ 1996; 313: 58386.
Bentwich Z, Kalinkovich A, Weisman Z. Immune activation is a
dominant factor in the pathogenesis of African AIDS. Immunol Today
1995; 16: 18791.

Department of Medical Ecology and Health Management,

Hebrew University-Hadassah School of Public Health, Jerusalem, Israel
(N Galai)

Isolation and partial molecular

characterisation of a strain of Ebola
virus during a recent epidemic of
viral haemorrhagic fever in Gabon
M C Georges-Courbot, C Y Lu, J Lansoud-Soukate, E Leroy,
S Baize

Between 1994 and 1996, three successive outbreaks of

haemorrhagic fever have occurred in the tropical forest
region of north-eastern Gabon with a high fatality rate (57%
in Minkouka, 66% in Mayibout1; final figures not yet
available for Boou). Data concerning the first two Gabonese
epidemics, as well as Ebola virus isolation carried out at
Centre International de Recherches Mdicales de Franceville
(CIRMF), have been previously reported.1 It is clear from
these and from results of serological surveys that the Ebola
virus is present in Gabon2 and widespread throughout
equatorial Africa.3
The third epidemic started on July 13, 1996, in Boou,
north-eastern Gabon. There have been no new cases
reported since Oct 30. 24 cases presenting with high fever,
diarrhoea, vomiting, and haemorrhagic symptoms have been
reported to date, one of which was a Gabonese doctor who
contracted the disease when treating a patient and who
subsequently sought medical care in South Africa, where it is
believed he contaminated the nursing personnel. Among
these confirmed cases, 17 have died; a 68% fatality rate. On
Oct 4, the Gabonese health authorities sent CIRMF sera
from two clinically confirmed cases and three exposed
individuals. These sera were tested by Ebola antigen virus
capture ELISA assay (using reagents kindly provided by the
Special Pathogens Branch, CDC, Atlanta, GA, USA). The
two clinical cases had high titres (16 and >256) of virus
antigen but the three others were negative. The five sera were
also screened by indirect immunofluorescence for antibodies
(IFA) to Crimean-Congo Fever, Rift Valley Fever, Ebola,
Lassa, and Marburg (CRELM test). All gave negative
results as did sera from the first Ebola epidemic in Gabon,
demonstrating the lack of sensitivity of IFA during the acute
phase of Ebola virus infection.
On Oct 27, sera from two other cases taken during the
acute symptomatic phase (titres >256 for Ebola virus
antigen) were used to inoculate Vero cell cultures. Infected
Vero cells showed cytopathogenic features 7 days after
inoculation with a high titre of viral antigen as shown by
immunocapture. In order to compare both strains we
extracted viral RNA from Gabon strain 1996/2 (Boou) and
1994 (Minkouka) from peripheral blood mononuclear cells
of patients and supernatants of Vero cultures with RNeasy kit
(Qiagen). The first strands of cDNA were synthesised with
GeneAmp RNA PCR kit (Perkin-Elmer). The amplification
was carried out using the DNA thermal cycler 480 (PerkinElmer) with degenerate primers from a conserved region of

Vol 349 January 18, 1997

the L gene, selected using sequences of the Sudan strain, and

two Marburg strains (GenBank data). The PCR products of
both 1994 and 1996/2 Gabon strains were directly sequenced
using Sequenase II kit (Amersham).
The Gabonese strains are very similar: in the first 156 bp
sequence, a single base (position 110, A>G substitution)
differentiated the two strains. This result indicates that Ebola
viruses circulating in Gabon were stable during these two
outbreaks which occurred at an 18-month interval.
Molecular characterisation of the nuclearprotein and
glycoprotein gene regions is in progress in our laboratory, as
well as comparison (on the basis of our first results) with
Ebola Zaire strain to which Ebola Gabon strains seem to
present high homology (>99%) compared with Sudan Maleo
strain (773%), Marburg Popp strain (713%), and Marburg
Musoke strain (694%).
1

Georges AJ, Renaut AA, Bertherat E, et al. Recent Ebola virus

outbreaks in Gabon from 1994 to 1996: epidemiologic and control
issues. Report of International Colloquium on Ebola virus research,
Antwerp, 1996.
Ivanoff B, Duquesnoy P, Languillat G, et al. Hemorrhagic fever in
Gabon: incidence of Lassa, Ebola and Marburg viruses in HautOgoou. Trans R Soc Trop Med Hyg 1982; 76: 71920.
Johnson E, Gonzalez JP, Georges AJ. Eilovirus activity among selected
ethnic groups inhabiting the tropical forest of equatorial Africa. Trans
R Soc Trop Med Hyg 1993; 87: 53638.

Unit de Biologie des Rtrovirus et des Pathognes viraux spciaux

(M-C Georges-Courbot), Unit de Gntique et de Pharmacogntique,
and Unit de Recherche sur les Maladies Emergentes Rmergentes,
Centre International de Recherches Mdicales de Franceville (CIRMF),
BP 769 Franceville, Gabon

Identification of the Ebola virus in

Gabon in 1994
Jacques Amblard, Paul Obiang, Samuel Edzang, Christophe
Prehaud, Michle Bouloy, Bernard L E Guenno

Gabon has been struck twice during 1996 by the Ebola virus.
One epidemic began in February in the village of Mayibout,
70 km north of Makokou, the main town of the OgoueIvindo province.1 Among the 37 cases, 21 died. The second
outbreak was from July to December around the town of
Boou, 200 km west of Makokou,2 and reached Libreville
and Johannesburg. It caused 52 cases and 40 deaths. We
report here the virological confirmation of a previous
outbreak of Ebola haemorrhagic fever in Gabon.
In November, 1994, unexplained deaths were reported in
two gold-mining camps, Mekouka and Andock. During
December, the number of cases increased in the two camps
and some appeared in the nearby village of Minkebe. These
three places are situated in deep rain forest along the Nouna
river, 100 km north-west of Makokou, and can be reached
only by canoe. A team from the Ministry of Health was sent
and blood samples collected in Makokou Hospital and in
Minkebe. The main clinical symptoms were fever, abdominal
pain, black diarrhoea, and conjunctival injection. Two
diseases were suspected, mercury poisoning and yellow fever.
The first eight samples from acutely ill patients were sent to
military hospitals in France. The concentrations of mercury
in sera were too low to support mercury poisoning. Six of
these samples were tested at the Pasteur Institute for yellow
fever IgM and found negative. A retrospective analysis
detected a high titre of Ebola IgM in four of them.
Unfortunately, the volumes were too low and the storage
conditions too bad to allow viral isolation.
A second set of samples had been collected in Minkebe on
Dec 27, 1994. Among 33 persons complaining of at least one

181

THE LANCET

symptom of fever, headache, or diarrhoea, two men (34 and

28 years old) had haemorrhagic fever. They had been
evacuated to Makokou where one died and the other
recovered. We isolated Ebola viruses from both sera.
Furthermore, Ebola specific IgM was detected in 9 sera
(27%). We have sequenced the whole nucleocapsid gene of
this Ebola Gabon 1994 strain and a part of the envelope
genes has been sequenced in Marburg (Heinz Feldmann,
Viktor Volchkov, personal communication). It is very closely
related to the Zaire strain isolated in 1976 (Yambuku) and
1995 (Kikwit) with less than 15% divergence in both
nucleotide sequences. The sequences of the PCR amplified
genes of the samples collected in February, and October,
1996, during the last outbreaks in Gabon are nearly identical
to this 1994 strain.
This epidemic has been primarily reported as a yellow
fever outbreak on serological results obtained on other
samples but without isolation of the virus.3 Sylvatic yellow
fever is often associated with mild forms of the disease or
may be symptomless.4 Here we have proof of the cocirculation of both viruses but the clinical symptoms and
virological results suggest that the 44 reported cases with 28
deaths5 were due to Ebola.
The impact of the 1994 outbreak had increased the
surveillance of unusual diseases in the Makokou region and
allowed a rapid recognition of the following epidemics. These
early warnings have probably limited the number of cases.
1
2
3
4
5

Anonymous. Outbreak of Ebola haemorrhagic fever in Gabon officially

declared over. Wkly Epidemiol Rec 1996; 71: 12526.
Anonymous. Ebola haemorrhagic fever. Wkly Epidemiol Rec 1996; 71:
320.
Anonymous. Yellow fever. Wkly Epidemiol Rec 1995; 70: 163
Monath T. In: Monath T, ed. The arboviruses, epidemiology and
ecology. Chapter 51, CRC Press, 1989, USA.
Anonymous. Yellow fever. Wkly Epidemiol Rec 1995; 71: 64.

Laboratoire des Bunyavirides and Collaborating center for arboviruses

and hemorrhagic fevers, Institut Pasteur, 28 rue de dr. Roux, 75724
Paris 15, France (B le Guenno); and Ministry of Public Health and
Population, Libreville, Gabon

Breath alcohol concentrations in

men 78 hours after prolonged,
heavy drinking: influence of habitual
alcohol intake
Neil R Wright

This study addresses two questions: what morning-after

breath alcohol concentrations result from heavy social
drinking sessions, and how are concentrations affected by
levels of habitual intake?
I asked 58 men aged 2050 to count drinks they consumed
at evening events where they anticipated drinking heavily.
Partners and friends helped verify quantities and the time
that drinking stopped. All men ate either shortly before or
during drinking. Morning alcohol concentrations were
recorded with a Lion Laboratories (Barry, Wales) SD400P
intoximeter by the author in their own homes 78 hours after
drinking stopped. Habitual alcohol intake was estimated by
4-week prospective drinking diaries or accounts of frequency
of drinking and quantity per session in a typical month.
Nobody exceeded the UK drink-drive limit (35 g/100
mL) on morning testing. 30 men recorded zero breath
alcohol concentrations, five of whom consumed more than
20 units. Heavier habitual intake was associated with higher
session intake and higher morning-after breath alcohol

182

Habitual intake
Units/week
n

Units in
session
mean
(range)

Length of
session
mean, h:min
(range)

Drinking
rate mean,
units/h
(range)

BrAC g/100 mL
mean (range)

Light 09
n=17
Moderate 1028
n=18
Heavy 2950
n=11
Very heavy >50
n=12

11
(617)
17
(1026)
21
(1427)
31
(1447)

4:15
(3:006:00)
5:15
(3:007:00)
6:00
(4:307:15)
6:30
(3:0010:00)

26
(2133)
32
(2250)
35
(2744)
48
(2553)

43
(021)
32
(015)
86
(026)
132
(029)

Significant differences in BrAC (Mann-Whitney U tests): moderate vs heavy p=0049;

light vs very heavy p=0043; moderate vs very heavy p=0008.

Characteristics of drinking sessions and breath alcohol

concentrations (BrAC) 78 hours after cessation of drinking in
men age 2050 with different levels of habitual alcohol intake

concentrations (table). However, within group variation was

wide: in the habitually heavy group one man recorded 26
g/100 mL after drinking 14 units in 4
hours, whilst another
had zero breath alcohol concentration after consuming 23
units in 5 hours. On morning breath testing, 44 of 58 men
said they had a hangover and felt unfit to drive; 22 had zero
breath alcohol concentrations.
This study suggests that healthy individuals found to have
breath alcohol concentrations in excess of the drink-drive
limit in the morning have either consumed extraordinary
amounts of alcohol, or have been drinking less than 78
hours before testing. Higher morning concentrations in
habitually heavy and very heavy drinkers suggests that
raised alcohol elimination rate due to regular heavy drinking1
is a lesser influence on morning breath alcohol concentration
than is the strong tendency for habitually heavy drinkers to
consume more alcohol than lighter habitual drinkers at social
events.
The literature on the timecourse of breath and blood
alcohol concentrations is dominated by laboratory-style,
single-dose experiments in fasted volunteers. They indicate
that alcohol is detectable 8 hours after drinking 6 units.2 The
application of such findings to breath alcohol concentrations
after social drinking is misleading. The need for research into
the kinetics of alcohol following prolonged drinking has been
highlighted.3 Alcohol metabolism during prolonged drinking
produces lower post-drinking concentrations than after
single-dose intake. Food decreases alcohol concentrations
through its effect on alcohol absorption,4 and through postabsorptive mechanisms.5 These downward influences operate
in drinking in natural surroundings and explain the
discrepancy between the findings presented here and breath
alcohol concentrations predicted by most other studies.
This sort of study, however, has the inherent problem of
poor recall accuracy for quantity drunk. Alcohol kinetics of
social drinking is an under-researched topic with important
implications for drink-drive policy.
1

2
3
4

Olsen H, Sakshaug J, Duckert F, Stromme JH, Morland J. Ethanol

elimination rates determined by breath analysis as a marker of recent
excessive ethanol consumption. Scand J Clin Lab Invest 1989; 49:
35965.
Saunders JB, Paton A. ABC of alcohol. BMJ 1981; 283: 138081.
Dubowski KM. Absorption, distribution and elimination of alcohol:
highway safety aspects. J Stud Alcohol 1985; 10 (suppl): 98108.
Von Wartburg J-P. Pharmacokinetics of alcohol. In: Crow KE,
Batt RD, eds. Human metabolism of alcohol, volume 1.
Pharmacokinetics, medicolegal aspects, and general interests.
Boca Raton, Florida: CRC Press, 1989: 2021.
Hahn RG, Norberg A, Gabrielsson J, Danielsson A, Jones AW. Eating
a meal increases the clearance of ethanol given by intravenous infusion.
Alcohol Alcohol 1994; 29 (6): 673477.

University of Leicester, Department of Psychiatry, Robert Kilpatrick

Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX,
UK

Vol 349 January 18, 1997