710

CONCISE COMMUNICATION

Continued Transmission of West Nile Virus to Humans in Southeastern

Romania, 1997–1998
Costin Cernescu,1 Nicolae-Ion Nedelcu,2 Gratiela
Tardei,1 Simona Ruta,1 and Theodore F. Tsai3
1
Institute of Virology, Romanian Academy, and 2Ministry of Health,
Bucharest, Romania; 3Division of Vectorborne Infectious Diseases,
Centers for Disease Control and Prevention, Fort Collins, Colorado

After an epidemic of West Nile (WN) virus neurologic infections in southeastern Romania
in 1996, human and animal surveillance were established to monitor continued transmission
of the virus. During 1997 and 1998, neurologic infections were diagnosed serologically as WN
encephalitis in 12 of 322 patients in 19 southeastern districts and in 1 of 75 Bucharest patients.
In addition, amid a countrywide epidemic of measles, the etiology of the febrile exanthem in
2 of 180 investigated cases was determined serologically to be WN fever; 1 case was complicated

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by hepatitis. Sentinel chickens placed in Bucharest seroconverted to WN virus during the
summer months, indicating their potential value in monitoring transmission. The continued
occurrence of sporadic WN infections in southeastern Romania in consecutive years after the
1996 epidemic is consistent with local enzootic transmission of the virus.

Mosquitoborne West Nile (WN) virus usually is an acute
self-limiting febrile illness with exanthem and myalgia. How-
ever, the illness may be complicated by central nervous system
infection, especially in the elderly, and rarely by hepatitis or
other organ involvement. An unprecedented epidemic of WN
encephalitis occurred in southeastern Romania in 1996, pro-
ducing 393 cases—principally in Bucharest and districts bor-
dering the lower Danube River [1]. Although the outbreak was
suspected to have been an extension of sylvatic transmission
from the delta, previous field investigations had not character-
ized a local viral transmission cycle, and the virus had never
been isolated in the country until the 1996 outbreak. The results
of surveillance maintained in 1997 and 1998 to monitor con-
tinued transmission of the disease are reported here.
Methods
In 1997, Romanian public health epidemiologists in 19 districts
were instructed to collect serum and cerebrospinal fluid (CSF) spec-
imens from patients presenting with aseptic meningitis or enceph-
alitis between April and November for serologic diagnosis of WN
virus infection. In addition, 160 sentinel chickens in Bucharest were
bled biweekly between 26 June and 13 August and again on 16
October 1997 to monitor enzootic viral transmission. Virus-specific
IgG was detected by indirect ELISA [2]. In 1998, specimens from
meningoencephalitis patients hospitalized between March and Sep-
Received 3 March 1999; revised 26 October 1999; electronically published
8 February 2000.
Reprints or correspondence (present address): Dr. Theodore F. Tsai, Led-
erle Vaccines, 401 N. Middletown Rd., Pearl River, NY 10965 (Tsait@
war.wyeth.com).
The Journal of Infectious Diseases 2000; 181:710–2
q 2000 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2000/18102-0040$02.00
tember in Bucharest only were referred for serologic testing. At the
end of the summer, acute samples from 180 patients hospitalized
with acute measles were tested retrospectively. Human specimens
were tested at the Institute of Virology by IgM capture and indirect
IgG ELISAs [1].
Results
In 1997, specimens from 322 patients, 119 from Bucharest,
were evaluated for WN virus antibodies. Twelve patients with
clinical meningoencephalitis and elevated IgM absorbance ra-
tios in serum or CSF samples were diagnosed with cases of
acute WN encephalitis. In the only fatal case, the serologic
diagnosis was based on tests of acute serum and CSF samples
only, but in the others, IgG conversions between acute- and
convalescent-phase serum samples also were demonstrated. The
district residences of WN encephalitis patients and those in
whom the diagnosis was excluded are shown in figure 1. Lab-
oratory-confirmed WN neurologic infections occurred between
13 July and 25 September with onset of 2 cases in July, 6 in
August, and 4 in September. Case-patients were 13–76 years
old (median, 45).
Seroconversions in sentinel chickens in Bucharest were dem-
onstrated in each of four consecutive biweekly bleedings from
26 June to 13 August with 23%, 26%, 40%, and 16% of birds
converting at each interval. No seroconversions were noted
again until the final bleeding on 16 October, when 13% of the
sentinels seroconverted.
In 1998, clinical samples from 75 patients in Bucharest hos-
pitalized with encephalitis (25), aseptic meningitis (36), and me-
ningoencephalitis (14) were submitted for serologic testing. One
patient was diagnosed as having acute WN encephalitis.
From May 1997 to September 1998, the country experienced
JID 2000;181 (February) West Nile Virus Transmission in Romania
Figure 1. Serologically confirmed cases of West Nile virus and other neurologic infections by district of residence and etiology, Romania, 1997.
its greatest measles epidemic in the postvaccine era: 120,000
cases were reported, principally among high school children
and young adults (14–25 years old). Serum samples from 180
hospitalized patients were tested for WN IgG and IgM anti-
bodies in parallel with confirmatory tests for measles. Two pa-
tients with a clinical diagnosis of measles without serologic
evidence of measles virus infection had elevations of WN virus-
specific IgM, indicating recent infection with that virus. The
first case patient, a 24-year-old woman whose onset of illness
was on 24 May, made an uneventful recovery. The second pa-
tient, a 19-year-old woman, developed an acute febrile illness
with exanthem on 2 June and was initially diagnosed with a
case of acute measles, but her illness was complicated by hep-
atitis and sustained elevated hepatic transaminases for 2 weeks
before clinical recovery.
Discussion
The sporadic cases of WN virus infections in Bucharest and
the lower Danube delta in 1997 and in Bucharest in 1998 are
persuasive evidence of ongoing virus transmission in south-
711
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eastern Romania. Compared with the 1996 epidemic, when 352
(80%) of 441 hospitalized encephalitis patients with adequate
clinical specimens were diagnosed with cases of WN neurologic
infection, the 12 patients with laboratory-confirmed cases in
1997 comprised only 4% of the patients referred for evaluation,
and in 1998 only 1% [1]. The epidemiologic and clinical criteria
for specimen referral were similar in all years, underscoring the
unusual pattern of viral transmission in 1996. Systematically
collected data from other years are unavailable; however, the
experience from the aftermath of the epidemic, when clinical
suspicion of the diagnosis might still have been high, suggests
that WN encephalitis occurs sporadically and with a relatively
low frequency in Bucharest and in the southeastern quadrant
of Romania. Sentinel chickens were posted in Bucharest rather
late, but by the first bleeding on 26 June, nearly one quarter
of the sentinels had seroconverted, preceding the onset of the
first case in a human in the city by 6 weeks; this indicates the
potential value of sentinel bird surveillance for forecasting fu-
ture epidemics.
During the course of the 1996 investigation, hospital-based
case finding focused on identifying neurologic infections and
712 Cernescu et al.
attempts to measure the full extent of the epidemic by identi-
fying patients with milder illnesses were unsuccessful because
of the relatively low infection rate and the nonspecific symp-
toms associated with uncomplicated WN fever. In previous re-
ports, WN fever has been characterized as an undifferentiated
self-limited febrile illness, often with exanthem, lymphadeno-
pathy, and polyarthralgias [3]. But illness complicated by fatal
hepatitis, pancreatitis, myocarditis, and neurologic infection
have been described [4–7]. The 2 patients hospitalized with a
clinical diagnosis of measles appear to have had cases of WN
fever with exanthem, complicated in the second patient by acute
hepatitis. The frequency of the latter complication is uncertain,
although in the 1998 Georgia outbreak, in which 624 febrile
exanthem case patients were investigated, 3 had fatal hepatitis
[7]. Hepatitis complicating measles appears to be even rarer. A
recent review found only 27 reported cases with hepatobiliary
involvement [8]. On the basis of the negative serologic findings
of acute measles infection and the presence of WN virus-specific
IgM antibodies, it seems likely that this was a case of WN
fever-associated hepatitis.
Sporadic cases and clusters of encephalitis cases occurring
during the summer and attributed to WN virus were previously
reported in areas of Romania, but some cases had epidemio-
logic features more consistent with tick-borne encephalitis, and
WN virus was not isolated, so the diagnosis remains unconfir-
med [9, 10]. Although reports and serosurveys reporting WN
HI antibodies in birds, mammals, and humans in the Danube
delta strongly suggested that the virus was transmitted locally,
a mechanism of enzootic transmission was never defined, and
periodic reintroductions of the virus from Africa by migratory
birds also seemed possible. Perennial transmission of the virus
in the Danube delta is plausible in view of the plentiful pop-
ulation of birds in that expanse of wetlands, and the continued
human infections in 2 consecutive years after the 1996 outbreak
further supports that likelihood. The chance downing of south-
ward migrating storks (Ciconia ciconia) as they transited Israel
in August and September 1998 and the isolation of WN virus
from moribund and dead animals provide additional evidence
of viral transmission in Europe, at least in areas where this
species summers or along its southern flyway [11]. This obser-
vation underscores the possibility of intercontinental virus
transfer between Europe and Africa in both directions.
WN virus has been isolated sporadically since 1962 from a
broad area of southern and central Europe, including Ukraine,
Belarus, and western Russia, and outbreaks affecting humans
and horses have been documented in France, Ukraine, and the
JID 2000;181 (February)
Volga delta of Russia [12]. In addition, there is serologic evi-
dence of human and animal infection elsewhere on the conti-
nent. In this decade, WN outbreaks have been reported in Tus-
cany, Italy (14 cases in horses), in Georgia (624 cases in
humans), in the Czech Republic (5 cases in humans), and in
Algeria and Morroco (16 cases in humans); in addition to the
epidemic and postepidemic cases in Romania, these outbreaks
indicate increased transmission or recognition of the disease in
southern Europe and neighboring areas [7, 12–15].
References
1. Tsai TF, Popovici F, Cernescu C, Campbell GC, Nedelcu NI, investigative
team. West Nile encephalitis epidemic in southeastern Romania. Lancet
1998; 352:767–71.
2. Olson JG, Scott TW, Lorenz LH, Hubbard JL. Enzyme immunoassay for
Downloaded from http://jid.oxfordjournals.org/ by guest on October 21, 2012
detection of antibodies against eastern equine encephalomyelitis virus in
sentinel chickens. J Clin Microbiol 1991; 29:1457–61.
3. Marberg K, Goldblum N, Stork V, Jasinska-Klingberg W, Klingberg M. The
natural history of West Nile fever. I. Clinical observations during an ep-
idemic in Israel. Am J Hyg 1956; 69:259–69.
4. George AJ, Lesbordes JL, Georges-Courbot MC, Meunier DMY, Gonzalez
JP. Fatal hepatitis from West Nile virus. Ann Inst Pasteur 1987; 138:
237–44.
5. Perelman A, Stern J. Acute pancreatitis in West Nile fever. Am J Trop Med
Hyg 1974; 23:1150–2.
6. Albagli C, Chaimoff R. A case of West Nile virus myocarditis. Harefuah
1959; 57:274.
7. Tsereteli DG, Tsiklauri RA, Ivanidze EA. West Nile fever in West Georgia
[abstract 60.006]. Program and abstracts: 8th International Congress on
Infectious Diseases, Boston, 1998:206.
8. Khatib R, Siddique M, Abbass M. Measles associated hepatobiliary disease:
an overview. Infection 1993; 21:112–4.
9. Draganescu N, Garjabu E, Iftimovici R, Klebleev E. Études sérologiques sur
la présence de quelques arbovirus dans la région de sylvo-steppe de Tulcea.
Rev Roum Virol 1991; 42:47.
10. Draganescu N, Girjabu E, Iftimovici R. Investigations sérologiques chez
l’homme et chez differentes espèces d’animaux domestiques de la région
de sylvo-steppe de Roumanie, pour le dépistage de quelques Togaviridae
et Bunyaviridae. Rev Roum Virol 1993; 44:207–10.
11. Malkinson M, Banet C, Weisman J, Pokamonski S, King R. West Nile fever;
recent evidence for intercontinental dispersion [abstract VW28,03]. Pro-
gram and abstracts: XI International Congress of Virology, Sydney, 1999:
56.
12. Hubalek Z, Halouzka J. West Nile fever, a reemerging mosquito-borne viral
disease in Europe. Emerg Infect Dis 1999; 5:643–50.
13. Le Guenno B, Bougermouh A, Azzam T, Bouakaz R. West Nile: a deadly
virus. Lancet 1996; 349:1315.
14. El Harrack M, LeGuenno B, Gouno P. Isolement du virus West Nile au
Maroc. Virologie 1997; 1:248–9.
15. Mitchell CJ. Geographic spread of Aedes albopictus and potential for in-
volvement in arbovirus cycles in the Mediterranean basin. J Vector Ecol
1995; 20:44–58.