Surveillance for West Nile, Dengue, and Chikungunya Virus Infections, Veneto Region, Italy, 2010

In 2010, in Veneto Region, Italy, surveillance of summer fevers was conducted to promptly identify autochthonous cases of West Nile fever and increase detection of imported dengue and chikungunya in travelers. Surveillance highlighted the need to modify case definitions, train physicians, and when a case is identified, implement vector control measures

were already included in regular surveillance, to acquire a more reliable picture of disease transmission in the region.

The Study
In accordance with the study protocol, possible cases detected by general physicians and emergency department physicians had to be referred within 24 hours to the closest Unit of Infectious or Tropical Diseases. Serum samples from persons with possible cases were sent to the regional reference laboratory (Padua, Italy) for confi rmation. If neuroinvasive disease was present, the specifi c protocol for WNND was followed (1).
We defi ned a possible case of DENV or CHIKV infection as fever >38°C during the past 7 days in a traveler who had returned within the previous 15 days from countries to which these viruses are endemic, absence of leucocytosis (leukocyte count <10,000 μL), and absence of other obvious causes of fever. After malaria was ruled out, cases were further classifi ed as probable if rapid tests yielded positive results for dengue and chikungunya viruses. Rapid tests included detection of anti-CHIKV IgM by using the OnSite Chikungunya IgM Combo Rapid Test (CTK Biotech, Inc., San Diego, CA, USA) and of DENV nonstructural protein (NS) 1 antigen by using the Dengue NS1 Ag STRIP (Bio-Rad Laboratories, Hercules, CA, USA) on serum samples. Samples from persons with possible cases were sent to the regional reference laboratory for second-line laboratory testing and confi rmation. Second-line laboratory testing consisted of detection of DENV and CHIKV nucleic acids in plasma specimens by using real-time PCR and endpoint PCR, respectively, and detection of serum IgM and IgG by using an anti-CHIKV indirect immunofl uorescence assay (Euroimmun AG, Lübeck, Germany), DENV IgG DxSelect (Focus Diagnostics, Cypress, CA, USA), and DENV IgM Capture DxSelect (Focus Diagnostics). Samples with DENV -positive results by ELISA were further tested by plaque-reduction neutralization test to confi rm specifi city of antibody response. Confi rmed cases were defi ned as the presence of viral nucleic acid in blood specimens or by seroconversion or detection of increasing serum levels of specifi c IgM and IgG. Possible autochthonous cases of WNF were defi ned as fever >38°C for <7 days, age >15 years, no recent travel history, rash, and absence of other obvious causes of fever ( Figure 1).
Four (11%) of 38 possible cases of autochthonous WNF were confi rmed. All were positive for WNV IgM and/or IgG and confi rmed by plaque-reduction neutralization test, but none were WNV RNA positive. Clinical descriptions of WNF and WNND cases are reported elsewhere (1).

Conclusions
DENV, CHIKV, and WNV infections are arboviral diseases that fi nd potentially suitable vectors in Italy, particularly in Veneto. No autochtonous case of fever caused by DENV has been documented in Italy, but the possible role of the Aedes albopictus mosquito as a vector has been demonstrated by recent cases in France (2) and Croatia (3).
CHIKV caused the well-known outbreak in Emilia Romagna Region (northern Italy) in 2007, which was detected, by coincidence a few days after the imported cases in Italy had been reported (4); the published report concluded that "the possibility of introducing CHIKV into Italy cannot be ruled out on the basis of current evidence." The index case had occurred ≈2 months before the fi rst case was diagnosed (5). The recent occurrence of 2 locally transmitted cases of chikungunya in France, despite a low number of imported cases (6), shows that the risk remains high.
Because we were concerned about being overwhelmed by an unmanageable number of case reports of unspecifi c fevers, we chose a selective case defi nition, particularly for WNF, with the obligatory presence of a rash, and thereby lowered the sensitivity of the surveillance. However, the proportion of virus-positive patients was strikingly high: ≈20% of persons tested who had imported fever were positive for DENV or CHIKV, as were 10% of persons with locally acquired fevers for WNV. Compared with the 2 previous years, the special surveillance enabled detection of substantially more cases, showing that you only fi nd what you are looking for (Table). WNV circulation has now been documented in many areas of Italy, from north to south, through retrospective screening of solid organ donors (12) and through entomologic (13) and animal surveillance (14); nevertheless, in 2010, no human clinical cases were detected outside Veneto.
The success of this pilot phase prompted regional authorities to propose a 3-year plan, which the Ministry of Health has approved and funded, as part of the integrated surveillance of arboviral diseases, along with animal and entomologic surveillance. Relying only on the latter 2 would not be sensible. However, mosquito surveillance was able to predict cases in animals and humans ( Figure 2). Expected rates of WNV infection in mosquitoes at the only site with repeated positivity in animals, humans, and vectors (Venice Province) are shown together with the time of exposure of animals and humans in the same province. Time of exposure was estimated as 1 week before onset of symptoms (incubation range 2-14 days) (15). When the expected rate of mosquito infection was low (i.e., 0.06%), no clinical cases were recorded; when the expected  rate of infection was higher (>0.24%), clinical cases were observed in animals and humans.
Concerning the new plan for human surveillance of summer fevers, the case defi nition, particularly for WNF, has been modifi ed by removing the compulsory presence of rash, to enhance sensitivity. Training and sensitization of general practitioners and emergency department physicians play a fundamental role. On the basis of a predefi ned threshold of vector intensity in an area where a new case has been identifi ed, immediate vector control measures will be started when necessary. Dr Gobbi is an infectious diseases consultant with at the Centre for Tropical Diseases of Sacro Cuore-Don Calabria Hospital, Negrar (Verona), Italy. His primary research interests are travel-related infectious diseases and rapid diagnostic tests for malaria.