Clin Microbiol Infect 2012; 18: E545–E547
In 2011, from 26 September to 16 October, a small outbreak of West Nile virus (WNV) disease occurred on the island of Sardinia (Italy). According to the national case definition, six cases with acute neurological disease were confirmed in hospitalized patients, and four of them died; one of these was only 34 years old. In two case, WNV RNA was detected in urine, suggesting renal involvement. Sequence analysis showed lineage 1 and 2 circulation.
West Nile virus (WNV) belongs to the Japanese encephalitis virus group. This flavivirus is widespread in Africa, Europe, the Middle East and parts of Asia, and it has been introduced into North America in the late 1990s . Its transmission cycle involves birds as vertebrate hosts, and ornithophilic mosquitoes as maintenance vectors. Less than 1% of WNV-infected humans develop acute neuroinvasive disease, including meningitis, encephalitis, and flaccid paralysis .
Evidence of persistent WNV infection was shown in experimentally infected monkeys and hamsters, where the virus may be detected in urine samples for up to 12 months [3,4]. These experimental data raise the possibility that persistent renal infection may occur in humans. To this purpose, WNV presence was demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) in the urine of a patient with acute and persistent WNV infection .
Isolates of WNV fall into two major genetic lineages: the lineage 1 identified in North America, North Africa, Europe and Australia; and lineage 2, which is endemic in Southern Africa and Madagascar [6,7]. In recent years, lineage 2 circulation has been identified in central and eastern Europe, both in animal and human outbreaks [8–10].
In Italy, the first outbreak of WNV infection was reported among horses in 1998 in Tuscany . Then, the virus re-emerged in 2008 in the north-east of the country, causing sporadic cases and/or clusters of West Nile neuroinvasive disease (WNND) in humans and horses every year [12,13]. All Italian cases were due to lineage 1. During the summer of 2011, WN virus lineage 2 has been detected in urine samples of a febrile patient in the Marche Region . Moreover, in 2011, evidence of circulation of both WNV lineage 1 and 2 (very close to the Hungarian one) was demonstrated in animals in the north-east of Italy, suggesting a probable introduction of lineage 2 from central and/or eastern European countries, possibly through migratory bird routes .
Here, we report a cluster of human cases of WNND that occurred in the Sardinia region at the end of the 2011 summer season.
Between 26 September and 16 October 2011, we received biological samples of nine hospitalized patients with suspected WNND. All patients were from Sardinia, eight of them from the southwestern province of Oristano and one from the northeastern province of Olbia. Six of them had central nervous system manifestations and four died. WNV infection was confirmed for four cases, while the other two cases were classified as probable cases (Table 1). All these individuals were males and their median age was 68 years (range, 34–83). The cluster was characterized by a high case-fatality rate (CFR) (three of the four confirmed WNND cases (75%) died), compared with that observed in Italy from 2008 to 2011 (16%, 7/43 confirmed WNND cases)  One death occurred in a 34-year-old man. Whether that high fatality rate was due to a high virulence of the circulating strains, increased host susceptibility due to unknown factors or an under-detection of milder cases remains undefined.
|S 58||76||Oristano||F – M||Confirmed Death||+||+||+||+||–||+||L1|
|S 59||79||Oristano||F – M||Confirmed Death||+||NA||+||–||NA||NA|
|S 60||34||Oristano||F – M||Confirmed Death||+||+||+||–||+||+||L1|
|S 70||66||Oristano||F – P||Probable||+||NA||+||–||NA||NA|
|S 73||71||Olbia||F – M||Confirmed||+||+||–||+||+||NA||L2|
|S 74||83||Oristano||F – M||Confirmed Death||+||+||ND||+||–||NA||L1|
West Nile virus IgM-capture ELISA assay (FOCUS Diagnostics) was performed for the detection of IgM antibodies against WNV in sera, plasma and CSF. Six sera and four CSF samples were IgM positive by ELISA. IgM positive diagnosis was confirmed by plaque reduction neutralization test (PRNT) conducted against lineage 1 virus, or by genome detection. In one case, PRNT was not performed due to the small volume of serum received; however, this case was confirmed by positive PCR results both in serum and CSF. In one case PRNT was found negative: in this case lineage 2 virus could be detected in serum (Table 1).
RT and hemi-nested PCR were performed to detect viral RNA in serum, plasma, urine and CSF samples .
Positive PCR results could be obtained in three out of nine serum samples, two out of four CSF samples and two out of two urine samples (Table 1).
The PCR products were sequenced and aligned with the sequences that showed higher percentage of identity (99–97%) after Blast analysis. For phylogenetic analysis the HKY+G evolutionary model was chosen as the best-fitting nucleotide-substitution model, according to the hierarchical likelihood-ratio test (LRT) implemented in the modeltest v. 3.7 software . Modeltest utilizes the PAUP software , under a strict maximum likelihood (ML) approach, to test 56 different models of nucleotide substitution on a given sequences alignment.
The ML tree (Fig. 1) showed a circulation of both WNV lineages, with the main cluster due to lineage 1, and one isolated case in the eastern province of Olbia due to lineage 2. The spread of these lineages was apparently restricted to two geographically separated areas of the island. In particular, sequences from three WNV-positive patients from Oristano (southwestern Sardinia) revealed a virus strain belonging to the genetic lineage 1 (Accession JX122763), related to the WNV strains circulating in Italy in the years 2008 and 2009 and to the strains circulating in Europe and Israel from late 2004 to 2011. A sequence from the WNV-positive patient coming from Olbia (north-eastern Sardinia) showed a virus strain belonging to the genetic lineage 2 (Accession JX122764). This strain was closely related to those responsible for the outbreaks that occurred in Greece and Hungary  in 2010 and 2005, respectively, and to the first autochthonous Italian human case found in 2011 in Ancona .
In conclusion, WNV infection appears to be established in the Mediterranean basin and every year tends to invade new areas. Both lineage 1 and lineage 2 strains circulate in Europe, including Italy. In the summer of 2011, weather conditions (frequent rainfalls, high temperatures and high relative humidity) were favourable to mosquito population increase. Culex pipiens, which was already identified as the vector of WNV during the 1998 outbreak in Tuscany , was probably the main vector of WNV infection in the Sardinia outbreak. This hypothesis is supported by the WNV isolation from Cx. pipiens pools collected in the province of Oristano during last summer .
Further studies are needed to clarify the pathogenic impact of different strains. Thus, integrated human and animal surveillance is important to monitor the viral spread. Enhanced vector mosquito control programmes, together with the implementation of blood transfusion measures, are necessary to prevent infection transmission.
Of the two cases for which urine samples were available, WNV could be detected in one case (S58) in urine and serum (9 days after onset) but not in CSF (4 days after onset), and in one case (S60) only in urine and CSF but not in serum 11 days after the onset of the symptoms. Recent studies detected WNV RNA in the urine of five out of 25 people (20%) tested several years after their initial acute WNV disease . Moreover, urine samples from the Marche Region WNV case were positive by RT PCR up to 25 days after the onset of the disease .
Collection and testing of urine samples might be considered in the national surveillance and control protocols, as well as for transplant donors. Thus, these data suggest that tests based on the detection of WNV RNA in serum and/or CSF may give false-negative results, due to the short duration of viraemia. The addition of WNV RNA testing in urine, as well as in plasma and CSF, in the work-up of patients with suspected WNV infection, may improve the sensitivity of molecular testing for the diagnosis of WNV infection.
The authors thank the staff at the Sardinia Sanitary Italy agencies for providing clinical specimens. We thank Dr Monica Meola for technical support.
This research was partially funded by EU grant HEALTH.2010.2.3.3-3 Project 261391 EuroWestNile.
The authors declare that they have no competing interests.