Outbreak of West Nile virus infection in Greece, 2010.

Abstract

During 2010, an outbreak of West Nile virus infection occurred in Greece. A total of 197 patients with neuroinvasive disease were reported, of whom 33 (17%) died. Advanced age and a history of heart disease were independently associated with death, emphasizing the need for prevention of this infection in persons with these risk factors.

Full text

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Outbreak of West
Nile V irus Infec tion
in Greec e, 2010
Kostas Danis, Anna Papa,
George Theocharopoulos, Georgios Dougas,
Maria Athanasiou, Marios Detsis, Agoritsa Baka,
Theodoros Lytras, Kassiani Mellou,
Stefanos Bonovas, and Takis Panagiotopoulos
During 2010, an outbreak of West Nile virus infection
occurred in Greece. A total of 197 patients with neuroinvasive
disease were reported, of whom 33 (17%) died. Advanced
age and a history of heart disease were independently
associated with death, emphasizing the need for prevention
of this infection in persons with these risk factors.
A
the summer of 2010. The fi rst cases were diagnosed and
reported to the Hellenic Centre for Disease Control and
Prevention (HCDCP) on August 5, 2010 (1). WNV lineage
2 sequences were later obtained from 3 pools of Culex
pipiens mosquitoes trapped at 2 sites where cases of West
Nile neuroinvasive disease (WNND) had occurred (2).
Human cases of WNV disease had not been previously
reported in Greece. Serosurveys in the early 1960s, 1980s,
and 2007 identifi ed WNV antibodies in ≈1% of the
population, suggesting that WNV, or a related fl avivirus, was
circulating in Greece (3–5). In contrast, during 2005–2007, a
total of 9,590 blood donors were tested by WNV nucleic acid
amplifi cation assay and results were negative (6).
n outbreak of West Nile virus (WNV) infection
occurred in Central Macedonia in northern Greece in
The Study
After the outbreak alert was issued in early August
2010, physicians in Greece were asked to report all cases
of WNV infection to HCDCP, according to the current
European Union case defi nition (1). Only deaths that
occurred during hospitalization were attributed to WNV
infection. Statistical methods are described in the online
Technical Appendix (www.cdc.gov/EID/content/17/10/11-
0525-Techapp.pdf).
Serum and cerebrospinal fl uid specimens were tested
for immunoglobulin (Ig) M and IgG against WNV by
using an ELISA (WNV IgM capture DxSelect and WNV
IgG DxSelect; Focus Diagnostics Inc., Cypress, CA, USA).
Titers >640 were obtained by microneutralization assay
against WNV in 14/14 patients who were positive for WNV.
Although tick-borne encephalitis virus (TBEV) and dengue
virus (DENV) are not prevalent in Greece, specimens
were also tested for TBEV and DENV by ELISA (TBE/
FSME IgM and TBE/FSME IgG; IBL International Gmbh,
Hamburg, Germany) and Dengue Virus IgM Capture Dx
Select and IgG Dx Select (Focus Diagnostics Inc.). All
specimens were negative for TBEV, and cross-reactivity
was seen with DENV, mainly for IgM (7).
Overall, 262 patients with WNV infection were
reported to HCDCP. Of these patients, 197 (75%) had
neuroinvasive disease (encephalitis, meningitis, or acute
fl accid paralysis), and 65 (25%) had WNV fever. This study
focused on patients with WNND, who were identifi ed and
reported more consistently because of disease severity.
Patient disease onset occurred within a 14-week
interval during July 6–October 5, and the outbreak peaked
in mid August (Figure 1). Most (94%) patients with WNND
were reported from the 7 districts of Central Macedonia
(Figure 2), and the epicenter of the outbreak was in Pella
and Imathia Districts.
Characteristics of patients with WNND are shown in
Table 1. Median age of patients with neuroinvasive disease
was 72 years (range 12–88 years). The attack rate for
WNND increased signifi cantly (p = 0.006) with age (Table
1). The incidence of WNND in older persons (>80 years
of age) was ≈50× higher than that among the youngest age
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1868 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 10, October 2011
Author affi liations: Hellenic Centre for Disease Control and
Prevention, Athens, Greece (K. Danis, G. Theocharopoulos, G.
Dougas, M. Athanasiou, M. Detsis, A. Baka, T. Lytras, K. Mellou, S.
Bonovas, T. Panagiotopoulos); Aristotle University of Thessaloniki,
Thessaloniki, Greece (A. Papa); and National School of Public
Health, Athens (T. Panagiotopoulos)
DOI: http://dx.doi.org/10.3201/eid1710.110525
Figure 1. Reported cases (n = 197) of West Nile neuroinvasive
disease, by week of symptom onset, Greece, July 6–October 5,
2010. *Excluding Thessaloniki.

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West Nile Virus Infection in Greece, 2010
group (<20 years of age). Persons living in rural areas were
2× as likely to show development of WNND than persons
living in urban areas (Table 1).
Encephalitis/meningoencephalitis (168 patients, 85%)
was the most prominent clinical syndrome among patients
with WNND, followed by meningitis (23, 12%). In addition,
10 (5%) patients with acute fl accid paralysis were reported, 6
(3%) of whom did not have meningitis or encephalitis.
A large proportion (74%) of patients with WNND
had >1 underlying chronic medical condition; the most
common were hypertension (39%), heart disease (24%),
diabetes mellitus (24%), and immunosuppression (10%).
Patients with WNND were 2× more likely (odds ratio 2.16,
95% confi dence interval 1.15–4.04) than patients without
WNND to have underlying conditions.
Thirty-fi ve patients died during hospitalization (33
had WNND), indicating an overall case-fatality rate of
17% among persons with WNND. Median age of WNND
patients who died was 78 years (range 49–87 years). The
case-fatality rate increased substantially (p<0.001) with
age (Table 2). Median interval from WNV disease onset to
death was 13 days (range 3–90 days). In 15 (45%) patients
with WNND who died, the interval between disease onset
and death exceeded 2 weeks.
WNND patients with >1 underlying disease were 5×
more likely to have died than patients without underlying
conditions. Those patients who had heart disease or a
stroke were ≈2.5× more likely to have died than patients
without these conditions. However, only older age and
heart disease were independent predictors of death in the
fi nal binomial regression model (Table 2). Supplementary
results are shown in the online Technical Appendix.
Conclusions
Human cases of WNV infection were detected in
several European and Mediterranean countries in 2010,
indicating an increased intensity of viral circulation (8).
Clinical cases of WNV infection in humans or animals
had not been previously reported in Greece. The present
outbreak was the largest in Europe since 1996, when a
large outbreak was observed in Romania (9). The outbreak
was located in Central Macedonia, which contains 90% of
the rice paddies and 70% of the wetland areas in Greece
and provides a favorable environment for reproduction
of mosquito vectors (8). The region also hosts one of
the largest populations of migratory birds in Greece.
Meteorologic data for the area indicate that 2010 was
warmer than previous years and unusually wet (8).
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 10, October 2011 1869
Figure 2. Incidence per 100,000 population of
197 reported cases of West Nile neuroinvasive
disease, by township of residence, Greece,
July–October 2010. Districts with >1 reported
neuroinvasive cases were divided into townships.
Dark black lines indicate borders of Central
Macedonia (north) and Thessalia (south).

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The overall case-fatality rate among patients with
WNND (17%) was higher in Greece than that in other
countries (9–11). The reasons for this fi nding are not
clear. Many factors may have played a role in differences
in the fatality rate. These factors include diagnosis and
surveillance bias for more severe cases, virus strain,
host susceptibility, age structure of the population, and
underlying conditions.
Recent studies on WNV lineage 2 suggested that this
virus may be underestimated as a cause of neuroinvasive
disease (2,12). WNV linage 2 isolated from Cx. pipiens
mosquitoes in the affected areas during this outbreak had
a nucleotide genetic similarity of 99.6% with the goshawk
Hungary 2004 strain (12). However, few severe cases of
WNV infection were reported in Hungary. Experimental
studies would verify whether the amino acid substitution
H249P detected in the Greek strain, which is a suspected
virulence marker in lineage 1 strains, is associated with
increased virulence (12).
Advanced age and heart disease were found to
independently predict the risk for WNND-related death.
The association between age and severe disease has been
reported (9–11). The contributing factor of age may relate
to a decrease in the integrity of the blood–brain barrier and
facilitate access of WNV to the central nervous system (13).
Heart disease, particularly cardiac arrhythmias, have also
been recognized as frequent contributors to death caused
by WNV encephalitis (13–15). Physiologic stress of WNV
infection may precipitate or exacerbate underlying medical
conditions resulting in death (14).
These fi ndings emphasize the need for primary
prevention of WNV infection in patients with these
predisposing conditions and close monitoring for cardiac
complications in elderly patients hospitalized with WNV
disease. Vector mosquito control programs, including
source reduction and larviciding of Culex spp. mosquitoes
and ongoing public health education and WNV surveillance
in disease-endemic and newly affected areas, remain the
cornerstones of WNV disease prevention and control.
Acknowledgments
We thank the physicians at the hospitals and local public
health authorities for providing assistance with surveillance
of WNV infections in Greece; personnel in the Reference
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1870 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 10, October 2011
Table 1. Characteristics of 197 patients with West Nile neuroinvasive disease. Greece, July–October 2010*
Characteristic No. patients
Age group, y
<20
20–29
30–39
40–49
50–59
60–69
70–79
?
>80
Sex
F
M
Place of residence
Urban
Rural
Districts in Central Macedonia
Chalkidiki
Thessaloniki
Pieria
Serres
Kilkis
Imathia
Pella
Total
Other districts (region)
Etoloakarnania (western Greece)
Kozani (Western Macedonia)
Kavala (Eastern Macedonia)
Larissa (Thessalia)
Total in Greece
*Incidence rates were calculated by using 2008 mid-year population estimates of the Hellenic Statistical Authority as the denominator. CI, confidence
interval; NA, not applicable.
Incidence per 100,000 population Risk ratio (95% CI)
4
3
6
9
0.18
0.20
0.34
0.55
1.27
2.44
8.01
9.63
Reference
1.08 (0.24–4.84)
1.87 (0.53–6.63)
3.03 (0.93–9.82)
6.93 (2.35–20.49)
13.31 (4.68–37.84)
43.74 (16.05–119.2)
52.62 (18.89–146.6)
18
29
85
43
88
109
1.59
1.97
Reference
1.26 (0.95–1.67)
110
87
1.38
2.92
Reference
2.12 (1.60–2.80)
4
60
9
21
12
39
41
186
3.99
5.27
7.02
11.15
13.92
27.06
28.26
15.00
0.76 (0.28– 2.09)
Reference
1.33 (0.66–2.69)
2.12 (1.29–3.48)
2.64 (1.42–4.91)
5.14 (3.43–7.69)
5.37 (3.61–7.98)
NA
1
1
1
8
0.46
0.65
0.71
2.80
1.76
0.09 (0.01–0.63)
0.12 (0.02–0.89)
0.14 (0.02–0.98)
0.53 (0.25–1.11)
NA197

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West Nile Virus Infection in Greece, 2010
Laboratory for Arboviruses for providing technical support;
personnel of HCDCP and the outbreak coordinating team for
organizing and implementing prevention and control program;
scientists and staff of Ecodevelopment SA for contributing to
mosquito control operations; and V. Sambri for performing
neutralization testing.
This study was supported by the HCDCP.
Dr Danis is a medical epidemiologist at the Offi ce of Vector-
borne Diseases and Zoonoses, Department of Surveillance,
Hellenic Centre for Disease Control and Prevention, Athens,
Greece. His research interests include epidemiology of vector-
borne and vaccine-preventable diseases and surveillance of
communicable diseases.
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Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 10, October 2011 1871
Table 2. Predictive factors of death for 197 patients with West Nile neuroinvasive disease analyzed by univariate and multivariate
analysis, Greece, July–October 2010*
Characteristic No. deaths, n = 33† Case-fatality rate, %
Age group, y
40–59 1§2.50
60–69 2 6.90
70–79 1517.65
?
>80
15 34.88
Sex
F 10 11.36
M 23 21.10
Underlying diseases
None 23.92
>1
3121.23
Hypertension
No 1915.70
Yes 14 18.42
Heart disease
No 1812.00
Yes 1532.61
Diabetes
No 2315.44
Yes 1020.83
Immunosuppression
No 31 17.42
Yes 2 10.53
Cancer
No 29 16.11
Yes 423.53
Stroke
No 28 15.30
Yes 5 35.71
Renal failure
No 31 16.40
Yes 2 25.00
*CI, confidence interval; NA, not applicable.
†Two additional patients with nonneuroinvasive disease died, and those deaths were not included in this analysis.
‡In logistic regression analysis, initial models included all variables for which the p value was <0.05 or the odds ratio was >1.1 or <0.90. Therefore, all
variables were included in the initial models. Variables were removed 1 at a time depending on results of statistical testing (p<0.05), by using the
likelihood-ratio test. All variables that remained significant in the final logistic regression model were included in the binomial regression model for the
estimation of adjusted risk ratios.
§Belonged to the 40–49-year age group.
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7.06 (0.97–51.59)
13.95 (1.93–100.9)
Reference
2.72 (0.26–28.40)
6.13 (0.83–45.17)
11.41 (1.56–83.52)
Reference
1.86 (0.93–3.69)
NA
NA
Reference
5.41 (1.34–21.82)
NA
NA
Reference
1.17 (0.63–2.20)
NA
NA
Reference
2.72 (1.49–4.95)
Reference
2.03 (1.14–3.64)
Reference
1.35 (0.69–2.63)
NA
NA
Reference
0.60 (0.16–2.33)
NA
NA
Reference
1.46 (0.58–3.66)
NA
NA
Reference
2.33 (1.07–5.10)
NA
NA
Reference
1.52 (0.44–5.28)
NA
NA

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Address for correspondence: Kostas Danis, Department of Surveillance
and Intervention, Hellenic Centre for Disease Prevention and Control,
Agrafon 3-5, Marousi, Athens 15123, Greece; email: daniscostas@yahoo.
com
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1872 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 10, October 2011
The opinions expressed by authors contributing to this
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the authors are affi liated.