Outbreak of West Nile Virus Infection in Greece, 2010

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.


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  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). The overall case-fatality rate among patients with WNND (17%) was higher in Greece than that in other countries (9)(10)(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)(14)(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. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 10, October 2011  *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. Incidence data were mapped by township and district (prefecture) by using GNU R software (www.gnu.org/s/r/).

Geographic Spread of the Outbreak
The first cases occurred in Central Macedonia in northern Greece. The outbreak evolved and peaked almost simultaneously in rural and urban parts of the region, including the city of Thessaloniki, the second largest city in Greece. Face-to-face interviews showed that that 17 of 29 patients with WNND among Thessaloniki residents had no travel history to another affected area, suggesting that transmission occurred in the city. Three weeks after the beginning of the outbreak, it spread southward to the adjacent Larissa district (Thessalia region). After the peak of the epidemic, sporadic cases occurred in 3 other regions, but there was no evident epidemiologic link with the other locations.

Hospitalization and Intensive Care Unit Admission
All but 1 of the reported patients with WNND were hospitalized, and 22 (12%) were admitted to an intensive care unit (ICU). The median length of stay in hospital for patients with WNND was 12 days (range 3-75 days) and was significantly (p = 0.034) higher than that for patients with non-neuroinvasive disease (9 days, range 3-39 days). Of all reported patients with non-neuroinvasive disease, 54 (83%) were hospitalized, 2 (3%) in an ICU. All 33 patients who died of WNND were hospitalized, of whom 13 (39%) were in an ICU.

Neurologic Manifestations and Age
The median age (74 years, range 19-88 years) of patients with encephalitis or meningoencephalitis was significantly higher (p<0.001) than that of patients with only meningitis (64 years, range 12-80 years). Thus, younger patients were more likely to have only meningitis, with the risk for meningitis decreasing by ≈4% (RR 0.96, 95% confidence interval 0.95-0.98) for every 10-year increase in age. The median age of patients with acute flaccid paralysis was 74 years (range 63-81 years).