VECTOR-BORNE AND ZOONOTIC DISEASES Volume 15, Number 4, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/vbz.2014.1711
West Nile Virus Outbreak in the Lombardy Region, Northern Italy, Summer 2013 Francesca Rovida,1 Antonella Sarasini,1 Giulia Campanini,1 Elena Percivalle,1 Giovanna Gorini,1 Bianca Mariani,1 Angelo Pan,2 Antonio Cuzzoli,3 Stefano Possenti,4 Lisa Manzini,5 Francesco Castelli,6 Nicola Bossini,7 Paolo Antonio Grossi,8 Concetta Castilletti,9 Mattia Calzolari,10 Davide Lelli,10 Alessandra Piatti,11 Fausto Baldanti,1 and the West Nile Virus Task Force
Abstract
In the summer of 2013, an outbreak of West Nile virus (WNV) infection occurred in the Lombardy, a region of northern Italy to the west of districts affected by WNV in previous years. Eighteen cases of human WNV infection were diagnosed—10 cases of acute WNV neuroinvasive disease and eight of WNV fever. In the same period, WNV was detected in birds (one crow) in horses (11 cases) and from mosquitoes (six pools). Key Words:
West Nile virus—Outbreak—Lombardy—Surveillance plan.
Introduction
W
est Nile virus (WNV) is a positive-sense RNA virus belonging to the Flaviviridae family (genus Flavivirus). It was isolated for the first time in 1937 from a febrile woman in the West Nile district of Uganda (Smithburn et al. 1940). The virus is now endemic in many countries and has caused large outbreaks in recent years in Europe and North America (Suthar at al. 2013). WNV is maintained in nature, cycling between birds, the main WNV vertebrate hosts, and mosquitoes. In particular, Culex spp. play a major role in transmission among vertebrates. Humans and horses are incidentally infected and represent ‘‘dead-end’’ hosts (Rossi et al. 2010). The majority (*80%) of WNV infections in humans are asymptomatic. Symptomatic infections are mostly characterized by a mild, self-limiting febrile illness (Rossi et al. 2010), whereas WNV neuroinvasive disease (WN-ND) develops in < 1% of WNV-infected persons. The elderly and immunocompromised patients are at higher risk
for WN-ND (Hayes et al. 2005), which can present as meningitis, encephalitis, or paralysis. Until the mid-1990s, WNV was responsible for sporadic infections in Europe (Barzon et al. 2013a). Since 1996, a large outbreak of WN-ND with a high fatality rate was reported in Romania, followed by other outbreaks and sporadic cases in the Czech Republic, southern Russia, and Hungary (Barzon et al. 2013a, Sambri et al. 2013). Over the last 15 years, outbreaks involving horses and humans have been reported in Portugal, Spain, France, Italy, and Greece (Zeller et al. 2010). Italy and Greece have been the most affected European countries in the last years, with human cases of WN-ND reported since 2008 in Italy and 2010 in Greece (Barzon et al. 2013a). In Italy, the first WNV outbreak was reported in 1998 from central Italy within Tuscany and only involved horses (Autorino et al. 2002). Thereafter, WNV infections apparently disappeared for almost 10 years, and it was again detected in horses in Emilia-Romagna in the summer of 2008 (Calistri et al. 2010). In the same year, the first human cases of WN-ND
1
Molecular Virology Unit, Microbiology and Virology Department, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy. Infectious and Tropical Diseases Unit, Istituti Ospitalieri di Cremona, Cremona, Italy. Emergency and Acceptance Department, Istituti Ospitalieri di Cremona, Cremona, Italy. 4 Nephrology and Dialysis Unit, Istituti Ospitalieri di Cremona, Cremona, Italy. 5 Infectious Diseases Unit, Azienda Ospedaliera Carlo Poma, Mantua, Italy. 6 Institute of Infectious and Tropical Diseases, University of Brescia, Brescia, Italy. 7 Transplantation Unit, Nephrology, Spedali Civili of Brescia, Brescia, Italy. 8 Infectious Diseases Unit, Department of Surgical and Morphological Sciences, University of Insubria, Varese, Italy. 9 Laboratory of Virology, National Institute for Infectious Diseases ‘‘Lazzaro Spallanzani’’, Roma, Italy. 10 Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna ‘Bruno Ubertini, Brescia, Italy. 11 Regional Health Authority of Lombardy, Milan, Italy. 2 3
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and WNV fever (WNF) were diagnosed in an area of the Po River Valley across the Emilia-Romagna and Veneto Regions, in northeastern Italy (Rossini et al. 2008, Barzon et al. 2009, Gobbi et al. 2009, Rizzo et al. 2012). In 2009, WNV infection remained restricted to northeastern Italy. In 2010, WNV activity decreased and human infections were detected primarily in the Veneto Region (Barzon et al. 2013a). In two subsequent years, the number of cases increased in the Veneto Region and the epidemics extended outside the Po River Valley following a northeastern direction involving the Friuli-Venezia Giulia Region at the border with Austria and Croatia (Barzon et al. 2013a), whereas clusters of WN-ND were also diagnosed on the island of Sardinia and one case of WN-ND was identified in southern Italy (Spissu et al. 2011, Barzon et al. 2013a). Until 2013, the Lombardy Region (20 million inhabitants) was only marginally involved in the WNV epidemics, when two human confirmed cases of WN-ND were reported in the southeastern province of Mantua bordering Emilia-Romagna Region in 2009 (Rizzo et al. 2012). In past Italian outbreaks, the succession of WNV lineage 1 and 2 strains phylogenetically related to the Mediterranean and Eastern European subtypes of lineage 1, clade 1a, or to the Hungarian-Greek lineage 2 strains has been shown (Barzon et al. 2013a). Following the first outbreak of WNV involving only horses (Central Italy, 1998) (Autorino et al. 2002), a national surveillance program supported by the Ministry of Health was established since 2002 to identify WNV circulation early. This multispecies surveillance was planned to screen wild birds, sentinel chickens, sentinel horses, equine neurological cases, mosquitoes, and humans (Calzolari et al. 2013). The surveillance plan has been updated annually from human and animal health authorities in line with changes in the WNV epidemiological situation (Angelini et al. 2009, Calzolari et al. 2011, Mulatti et al. 2013). In this report, an outbreak of WNV involving humans, mosquitoes, birds, and horses in four different provinces of the Lombardy Region is described, documenting the enlargement of the WNV endemic area in northern Italy. Methods European Union definition of human WNV infection
In 2008, the European Union established the clinical and diagnostic criteria for probable and confirmed WNV case definition (European Centre for Disease Prevention and Control [ECDC]). A case is considered probable if the patient meets any of the following clinical criteria—suggestive clinical presentation (encephalitis, meningitis, fever ‡ 38C) in the presence of WNV-specific immunoglobulin M (IgM) or IgG in serum with IgG seroconversion, or a four-fold increase in IgG titer on two subsequent samples. A confirmed case is defined as meeting the previous clinical criteria and one or more of the following additional criteria: (1) Isolation of WNV from blood or cerebrospinal fluid (CSF), (2) detection of WNV RNA in blood and/or CSF, (3) WNV-specific IgM in CSF, and (4) detection of WNV IgM at a high titer and detection of WNV IgG, confirmed by a neutralization assay. Laboratory investigation
For this study, serum and CSF samples of patients with potential WNV infections were tested for the presence of
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specific IgM and IgG antibodies (WNV IgM Capture DxSelect and WNV IgG DxSelect by Focus Diagnostics, Cypress, USA). Furthermore, the presence of WNV-specific antibodies was confirmed by a plaque-reduction neutralization test (PRNT) (Barzon et al. 2013b). Serum, CSF, and urine samples, collected during the acute phase, were examined for the presence of WNV RNA with three methods— a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) targeting a conserved region of WNV lineages 1 and 2 (Linke et al. 2007) and two pan-Flavivirus nested RTPCRs (Scaramozzino et al. 2001, Sa´nchez-Seco et al. 2005) followed by sequencing of amplicons. In addition, WNV RNA was searched for in urine samples as previously suggested (Barzon et al. 2013b). Furthermore, 455 stored CSF samples collected in six previous spring–autumn seasons, 2007–2012, which had been previously scored as negative for bacterial and virus infections, were investigated retrospectively to evaluate the potential unreported presence of WNV infection in Lombardy. Entomological and veterinary surveillance of WNV infections
From July to October, 2013, national and regional institutions implemented the entomological and veterinary surveillance program related to WNV in the Lombardy Region. The entomological surveillance is based on a range of collection sites placed either in at-risk areas or in surrounding areas and aims at identifying possible WNV vector species as well as at determining their abundance and distribution. Entomological sampling was performed in four sites from the beginning of the season, whereas 23 sites were additionally activated from the beginning of September in the frame of a regional surveillance plan. Mosquitoes were collected by attractive traps, grouped in pools according to species, data, and sites of sampling, and screened by molecular tests for the presence of WNV (Calzolari et al. 2013). A total of more than 20,000 mosquitoes were collected. The veterinary surveillance system was based upon testing of sentinel birds and horses with clinical signs of potential WNV infection. In the period July–October of 2013, 632 wild birds were sampled and tested by a WNV-specific molecular test (Tang et al. 2006). Ethics statement
The local Ethics Committee consent was not required because according to a Regional Surveillance and Preparedness Plan (DGR 12591, December 27, 2012), diagnostic detection of WNV infections in the Lombardy Region was centralized at the Regional Reference Laboratory (Molecular Virology Unit, Fondazione IRCCS Policlinico San Matteo, Pavia). Informed consent was not necessary because patients with suspected WNV infections were included in a Regional diagnostic protocol. Prospective samples (serum, CSF, and urine) were collected by clinicians and handled by Molecular Virology Unit personnel; data were analyzed anonymously according to a Regional Surveillance and Preparedness Plan (DGR 12591, December 27, 2012). The 455 control CSF samples were collected and stored during the period 2007– 2012 from as many hospitalized patients with neurologic disorders as surplus volume following completion of routine diagnostic assays. All samples had been previously scored as
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negative for bacterial and virus infections and were investigated retrospectively to evaluate the potential unreported presence of WNV infection in Lombardy. This retrospective analysis was performed according to guidelines of the Institutional Review Board of the Fondazione IRCCS Policlinico San Matteo on the use of biologic specimens for scientific purpose in keeping with Italian law (art.13 D.Lgs 196/2003). The personnel of Molecular Virology Unit participated in the collection of these samples, which were anonymized upon collection. Results
As shown in Table 1, in the period from August 13 to October 4, 2013, 10 confirmed cases of acute WN-ND and eight cases of acute WNF (six confirmed and two probable) were diagnosed. In Figure 1A, the weekly distribution of WNV cases is shown. WNV cases were distributed in four different provinces—[]]Cremona (n = 9), Mantua (n = 5), Brescia (n = 3), and Lodi (n = 1)—in the southeastern area of the Lombardy Region, in close proximity to areas of the Emilia-Romagna and Veneto Regions affected by WNV outbreaks in previous years (Fig. 1B). Among patients with WN-ND, nine (90%) were males and one (10%) female, with a median age of 75 years (range, 54– 89). WN-ND cases were detected in all the four provinces involved in the WNV outbreak (Table 1). All WN-ND patients required hospitalization: Eight presented with encephalitis, three of them died, and two presented with meningoencephalitis. WNV-specific IgM antibodies were detected in all serum samples and in seven CSF specimens.
WNV-specific IgG antibodies were detected in 10 serum samples and in four CSF. In nine WN-ND cases, the specificity of the WNV antibodies was confirmed by the neutralization test, whereas in one case the assay was not performed (Table 1). WNV RNA was detected in the serum sample of one patient and in the CSF and serum of another patient. WNV viruria was observed in a WN-ND patient, in the absence of CSF and serum positivity for WNV RNA (Table 1). Of the eight patients with WNF, four (50%) were males and four (50%) females, with a median age of 58 years (range, 17–87). The cases were mostly detected in patients living in the province of Cremona (n = 7) and one in the province of Mantua. Six were confirmed cases of WNF and two were probable. The most common clinical signs were fever (n = 8) and rash (n = 2). WNV-specific IgM antibodies were detected in all eight serum samples, whereas WNVspecific IgG antibodies were detected in six serum samples. The specificity of the WNV antibodies was confirmed by the neutralization test in six cases, whereas in two cases the assay was not performed (Table 1). WNV RNA was detected in the urine of a single patient (Table 1). Sequencing of the NS5 gene showed that all WNV strains belonged to lineage 2
Evidence of this outbreak during the summer of 2013 is further strengthened by the finding that among the 455 CSF samples collected during the six previous summer seasons (2007–2012) and tested for the presence of specific IgM and IgG antibodies and WNV RNA; only one was scored as IgG positive.
Table 1. Characteristics of Human WNV Infections, in the Lombardy Region, August–October, 2013 Age/ Patient sex 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 a
78/M 66/M 89/M 61/M 79/M 49/M 55/F 75/F 54/M 17/M 71/F 63/M 27/F 87/F 54/M 78/M 76/M 57/M
Origin
Clinical presentation
Mantua Mantua Mantua Mantua Mantua Cremona Cremona Cremona Cremona Cremona Cremona Cremona Cremona Cremona Brescia Brescia Brescia Lodi
Encephalitis Meningoencephalitis Encephalitis West Nile fever Encephalitis West Nile fever West Nile fever Encephalitis Encephalitis West Nile fever West Nile fever West Nile fever West Nile fever West Nile fever Encephalitis Encephalitis Meningoencephalitis Encephalitis
ELISA IgMa ELISA IgGb
RT-PCRc,d
Outcome Serum CSF Serum CSF Neutralization Serum CSF Urine Alive Alive Dead Alive Dead Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Dead
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1*f 1 1 1
1 1 1 NA 1 NA NA 1 1 NA NA NA NA NA -f 1 NA NA
1 1 1 1 1 1 1 1 1 1 1 1 1*f 1 1 1
1 NA 1 NA NA 1 NA NA NA NA NA -f 1 NA NA
1 1 1 ND 1 1 ND 1 1 1 1 1 1 1 ND 1 1 1
1 -e -
2 NA NA NA NA NA NA NA NA +e NA NA
NA NA NA 1 NA NA NA NA 1 +e NA -
WNV IgM Capture DxSelect (Focus Diagnostics). WNV IgG DxSelect (Focus Diagnostics). c Real-time RT-PCR WNV L1-2 (Linke et al 2007). d Nested RT-PCR pan-Flavivirus (Scaramozzino et al. 2001, Sa´nchez-Seco et al. 2005). e Real-time RT-PCR Flavivirus (Moureau et al. 2007). f WNV IgG/IgM IIFT (Euroimmun); *Convalescent serum sample. N WNV, West Nile virus; ELISA, enzyme-linked immunosorbent assay; IgM, immunoglobulin M; CSF, cerebrospinal fluid; M, male; F, female; NA, not available; ND, not done. b
WEST NILE OUTBREAK IN LOMBARDY
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FIG. 1. (A) Human cases of West Nile virus (WNV) infection by week of symptom onset, Lombardy Region, August– October, 2013. (B) Distribution of human and animal cases of WNV infection, Lombardy Region, summer, 2013. WNND, WNV neuroinvasive disease. Color images available online at www.liebertpub.com/vbz
The veterinary and entomological surveillance programs to investigate WNV in the Lombardy Region revealed the presence of six WNV positive pools of Culex pipiens, the main vector of WNV in Italy, in the provinces of Cremona (n = 2), Mantua (n = 2), and Brescia (n = 2) (Fig. 1B, Tables 2 and 3). Eleven infected horses were detected in province of Mantua (n = 3) and Cremona (n = 8) (Fig. 1B, Table 2). An
infected crow was detected in the province of Cremona (Fig. 1B, Table 2). Discussion and Conclusion
WNV was detected for the first time in the Lombardy Region in September, 2009, with two confirmed cases of
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Table 2. WNV Positivity in the Provinces of Lombardy Region during Veterinary Surveillance in 2013 Province
Cases
Date
Species
Mantua
3
September 02 August 28 August 06 September 23 August 09 August 25 September 18 October 07 August 08
Horses (seroconversion) Insects
2 Cremona
4
4 2 Brescia
1 2
fection prior to 2013 in Lombardy (http://sorveglianza.izs.it/ emergenze/west_nile/emergenze.html). As previously shown (Calzolari et al. 2013), the veterinary and entomological surveillance plans allow for detecting WNV circulation much earlier than the occurrence of the human cases and provide important epidemiological information on the circulating strains in a certain area that could infect humans. Having such a rapid health alarm system in place is of extreme value because it enables regional infrastructures for optimal management of acute human cases and adoption of prevention strategies. Sequencing of NS5 amplicons in two human cases of WNND showed > 95% nucleotide identity with WNV Rovigo strains (lineage 2) circulating in the Veneto Region in 2013 (GenBank acc. nos. KF647251.1 and KF5883365.1). Finally, our data support the diagnostic value of WNV detection in urine samples of infected patients (Barzon et al. 2013b) and stress the importance of its routine use. In conclusion, an outbreak of WNV in the summer of 2013 involving the southeastern provinces of the Lombardy Region is described as a part of the larger outbreak involving the neighboring Emilia-Romagna and Veneto Regions. This is the largest outbreak of WNV reported in Lombardy so far, showing a west bound spread of the virus after 5 years spread in a northeastern direction.
Horses (with clinical signs) Horses (seroconversion) Insects
July 04 September 06 August 29 September 05 September 19
Crow Insects
WN-ND in the province of Mantua (Rizzo et al. 2012). A seroprevalence study to detect anti-WNV–specific antibodies was carried out in blood donations in the metropolitan area of Milan in the summers of 2009 and 2011; no positive sera were found in 2009, whereas five positive sera were detected in 2011 (Gaibani et al. 2013). These data, along with an IgGpositive CSF sample in our retrospective series, would suggest the presence of individuals with subclinical WNV infection in Lombardy (Gaibani et al. 2013). However, the area where the infection was acquired could not be determined and it cannot be excluded that it was acquired when traveling in surrounding regions affected by the epidemics since 2008. On the other hand, the substantial absence of WNV-positive CSF over a period of 6 years would suggest that in Lombardy the virus was not actively circulating before the summer of 2013. The veterinary and entomological surveillance programs for WNV confirmed the presence of WNV infection in mosquitoes, horses, and birds in Lombardy in summer of 2013, further supporting the timing of the WNV outbreak in this region. Indeed, the surveillance programs conducted in horses and wild birds since 2009 confirm the absence of in-
Acknowledgments
We thank all of the technical staff for handling the specimens and performing the assays. We thank Daniela Sartori for manuscript editing and Laurence Kelly for English language revision. This work was supported by a grant from the Regional Health Authority of Lombardy, Milan, Italy (DGR no. 12591 of December 27, 2012). Members of the West Nile Virus Task Force are as follows: Virology Units—city and participants: Pavia (Fausto Baldanti, Francesca Rovida, Antonella Sarasini, Giulia Campanini, Elena Percivalle, Giovanna Gorini, Bianca Mariani); Roma (Maria Rosaria Capobianchi, Concetta Castilletti). Infectious and Tropical Diseases Unit—city and participants: Cremona (Angelo Pan, Fabio Zacchi); Brescia (Francesco Castelli). Infectious Diseases Unit—city and participants: Mantova (Paolo Costa, Lisa Manzini); Varese (Paolo Antonio Grossi).
Table 3. Mosquitoes Collected in Lombardy Region During Entomological Surveillance in (July–October, 2013) Brescia Species Aedes (Ae.) albopictus Ae. caspius Ae. cinereus Ae. geniculatus Ae. vexans Aedes spp. Anopheles maculipennis Culiseta annulata Culex (Cx.) modestus Cx. pipiens Total
n
Cremona
Pools
n
Lodi
Mantua
Total
Pools
n
Pools
n
Pools
n
Pools
n
Pools
3 2475 12 3 2249 1 1940 6 30 6752 13,471
3 30 3 2 30 1 21 6 7 51 154
121 2744 12 3 2368 7 1959 9 52 13527 20,802
37 42 3 2 38 3 25 8 10 125 293
8 1
6 1
68
11
26 6
10 2
16 262
7 9
2 6
1 2
7
3
3
1
107
3
1
1
2 15 3988 4022
1 2 20 33
193 229
13 27
18 1 7 580 991
3 1 1 19 43
2014 2089
Pavia
22 36
WEST NILE OUTBREAK IN LOMBARDY
Emergency Unit—city and participants: Cremona (Antonio Cuzzoli). Nephrology and Dialysis Unit—city and participants: Cremona (Stefano Possenti). Transplantation Unit, Nephrology—city and participants: Brescia (Nicola Bossini). Veterinary Unit—city and participants: Brescia (Mattia Calzolari, Davide Lelli, Antonio Lavazza). Regional Health Authority of Lombardy (Maria Gramegna, Alessandra Piatti, Piero Frazzi). Author Disclosure Statement
No competing financial interests exist. References
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Moureau G, Temmam S, Gonzalez JP, Charrel RN, et al. A realtime RT-PCR method for the universal detection and identification of Flaviviruses. Vector Borne Zoonotic Dis 2007; 7:467–477. Mulatti P, Bonfanti L, Capelli G, Capello K, et al. West Nile virus in north-eastern Italy, 2011: Entomological and equine IgM-based surveillance to detect active virus circulation. Zoonoses Public Health 2013; 60:375–382. Rizzo C, Salcuni P, Nicoletti L, Ciufolini MG, et al. Epidemiological surveillance of West Nile neuroinvasive disease in Italy, 2008 to 2011. Euro Surveill 2012; 17:pii=20172. Rossi SL, Ross TM, Evans JD. West Nile virus. Clin Lab Med 2010; 30:47–65. Rossini G, Cavrini F, Pierro A, Macini P, et al. First human case of West Nile virus neuroinvasive infection in Italy, September 2008—case report. Euro Surveill 2008; 13:pii=19002. Sambri V, Capobianchi M, Charrel R, Fyodorova M, et al. West Nile virus in Europe: Emergence, epidemiology, diagnosis, treatment and prevention. Clin Microbiol Infect 2013; 19:699–704. Sa´nchez-Seco MP, Rosario D, Domingo C, Herna´ndez L, et al. Generic RT-nested-PCR for detection of flaviviruses using degenerated primers and internal control followed by sequencing for specific identification. J Virol Methods 2005; 126:101–109. Scaramozzino N, Crance JM, Jouan A, DeBriel DA, et al. Comparison of Flavivirus universal primer pairs and development of a rapid, highly sensitive heminested reverse transcription-PCR assay for detection of Flavivirus targeted to a conserved region of the NS5 gene sequence. J Clin Microbiol 2001; 39:1922–1927. Smithburn KC, Hughes TP, Burke AW, Paul JH. A neurotropic virus isolated from the blood of a native of Uganda. Am J Trop Med Hyg 1940; 20:471–492. Spissu N, Panichi G, Montisci A, Fiore F. West Nile Virus outbreak in Sardinia, Italy, in 2011. J Infect Dev Ctries 2013; 7:6–9. Suthar MS, Diamond MS, Gale M. West Nile virus infection and immunity. Nat Rev Microbiol 2013; 11:115–128. Tang Y, Hapip CA, Liu B, Fang CT. Highly sensitive TaqMan RT-PCR assay for detection and quantification of both lineages of West Nile virus RNA. J Clin Virol 2006; 36:177–182. Zeller H, Lenglet A, Van Bortel W. West Nile virus: The need to strengthen preparedness in Europe. Euro Surveill 2010; 15:pii = 19647.
Address correspondence to: Fausto Baldanti Molecular Virology Unit Microbiology and Virology Department Fondazione IRCCS Policlinico San Matteo 27100 Pavia Italy E-mail: [email protected]
West Nile Virus Outbreak in the Lombardy Region, Northern Italy, Summer 2013 Francesca Rovida,1 Antonella Sarasini,1 Giulia Campanini,1 Elena Percivalle,1 Giovanna Gorini,1 Bianca Mariani,1 Angelo Pan,2 Antonio Cuzzoli,3 Stefano Possenti,4 Lisa Manzini,5 Francesco Castelli,6 Nicola Bossini,7 Paolo Antonio Grossi,8 Concetta Castilletti,9 Mattia Calzolari,10 Davide Lelli,10 Alessandra Piatti,11 Fausto Baldanti,1 and the West Nile Virus Task Force
Abstract
In the summer of 2013, an outbreak of West Nile virus (WNV) infection occurred in the Lombardy, a region of northern Italy to the west of districts affected by WNV in previous years. Eighteen cases of human WNV infection were diagnosed—10 cases of acute WNV neuroinvasive disease and eight of WNV fever. In the same period, WNV was detected in birds (one crow) in horses (11 cases) and from mosquitoes (six pools). Key Words:
West Nile virus—Outbreak—Lombardy—Surveillance plan.
Introduction
W
est Nile virus (WNV) is a positive-sense RNA virus belonging to the Flaviviridae family (genus Flavivirus). It was isolated for the first time in 1937 from a febrile woman in the West Nile district of Uganda (Smithburn et al. 1940). The virus is now endemic in many countries and has caused large outbreaks in recent years in Europe and North America (Suthar at al. 2013). WNV is maintained in nature, cycling between birds, the main WNV vertebrate hosts, and mosquitoes. In particular, Culex spp. play a major role in transmission among vertebrates. Humans and horses are incidentally infected and represent ‘‘dead-end’’ hosts (Rossi et al. 2010). The majority (*80%) of WNV infections in humans are asymptomatic. Symptomatic infections are mostly characterized by a mild, self-limiting febrile illness (Rossi et al. 2010), whereas WNV neuroinvasive disease (WN-ND) develops in < 1% of WNV-infected persons. The elderly and immunocompromised patients are at higher risk
for WN-ND (Hayes et al. 2005), which can present as meningitis, encephalitis, or paralysis. Until the mid-1990s, WNV was responsible for sporadic infections in Europe (Barzon et al. 2013a). Since 1996, a large outbreak of WN-ND with a high fatality rate was reported in Romania, followed by other outbreaks and sporadic cases in the Czech Republic, southern Russia, and Hungary (Barzon et al. 2013a, Sambri et al. 2013). Over the last 15 years, outbreaks involving horses and humans have been reported in Portugal, Spain, France, Italy, and Greece (Zeller et al. 2010). Italy and Greece have been the most affected European countries in the last years, with human cases of WN-ND reported since 2008 in Italy and 2010 in Greece (Barzon et al. 2013a). In Italy, the first WNV outbreak was reported in 1998 from central Italy within Tuscany and only involved horses (Autorino et al. 2002). Thereafter, WNV infections apparently disappeared for almost 10 years, and it was again detected in horses in Emilia-Romagna in the summer of 2008 (Calistri et al. 2010). In the same year, the first human cases of WN-ND
1
Molecular Virology Unit, Microbiology and Virology Department, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy. Infectious and Tropical Diseases Unit, Istituti Ospitalieri di Cremona, Cremona, Italy. Emergency and Acceptance Department, Istituti Ospitalieri di Cremona, Cremona, Italy. 4 Nephrology and Dialysis Unit, Istituti Ospitalieri di Cremona, Cremona, Italy. 5 Infectious Diseases Unit, Azienda Ospedaliera Carlo Poma, Mantua, Italy. 6 Institute of Infectious and Tropical Diseases, University of Brescia, Brescia, Italy. 7 Transplantation Unit, Nephrology, Spedali Civili of Brescia, Brescia, Italy. 8 Infectious Diseases Unit, Department of Surgical and Morphological Sciences, University of Insubria, Varese, Italy. 9 Laboratory of Virology, National Institute for Infectious Diseases ‘‘Lazzaro Spallanzani’’, Roma, Italy. 10 Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna ‘Bruno Ubertini, Brescia, Italy. 11 Regional Health Authority of Lombardy, Milan, Italy. 2 3
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and WNV fever (WNF) were diagnosed in an area of the Po River Valley across the Emilia-Romagna and Veneto Regions, in northeastern Italy (Rossini et al. 2008, Barzon et al. 2009, Gobbi et al. 2009, Rizzo et al. 2012). In 2009, WNV infection remained restricted to northeastern Italy. In 2010, WNV activity decreased and human infections were detected primarily in the Veneto Region (Barzon et al. 2013a). In two subsequent years, the number of cases increased in the Veneto Region and the epidemics extended outside the Po River Valley following a northeastern direction involving the Friuli-Venezia Giulia Region at the border with Austria and Croatia (Barzon et al. 2013a), whereas clusters of WN-ND were also diagnosed on the island of Sardinia and one case of WN-ND was identified in southern Italy (Spissu et al. 2011, Barzon et al. 2013a). Until 2013, the Lombardy Region (20 million inhabitants) was only marginally involved in the WNV epidemics, when two human confirmed cases of WN-ND were reported in the southeastern province of Mantua bordering Emilia-Romagna Region in 2009 (Rizzo et al. 2012). In past Italian outbreaks, the succession of WNV lineage 1 and 2 strains phylogenetically related to the Mediterranean and Eastern European subtypes of lineage 1, clade 1a, or to the Hungarian-Greek lineage 2 strains has been shown (Barzon et al. 2013a). Following the first outbreak of WNV involving only horses (Central Italy, 1998) (Autorino et al. 2002), a national surveillance program supported by the Ministry of Health was established since 2002 to identify WNV circulation early. This multispecies surveillance was planned to screen wild birds, sentinel chickens, sentinel horses, equine neurological cases, mosquitoes, and humans (Calzolari et al. 2013). The surveillance plan has been updated annually from human and animal health authorities in line with changes in the WNV epidemiological situation (Angelini et al. 2009, Calzolari et al. 2011, Mulatti et al. 2013). In this report, an outbreak of WNV involving humans, mosquitoes, birds, and horses in four different provinces of the Lombardy Region is described, documenting the enlargement of the WNV endemic area in northern Italy. Methods European Union definition of human WNV infection
In 2008, the European Union established the clinical and diagnostic criteria for probable and confirmed WNV case definition (European Centre for Disease Prevention and Control [ECDC]). A case is considered probable if the patient meets any of the following clinical criteria—suggestive clinical presentation (encephalitis, meningitis, fever ‡ 38C) in the presence of WNV-specific immunoglobulin M (IgM) or IgG in serum with IgG seroconversion, or a four-fold increase in IgG titer on two subsequent samples. A confirmed case is defined as meeting the previous clinical criteria and one or more of the following additional criteria: (1) Isolation of WNV from blood or cerebrospinal fluid (CSF), (2) detection of WNV RNA in blood and/or CSF, (3) WNV-specific IgM in CSF, and (4) detection of WNV IgM at a high titer and detection of WNV IgG, confirmed by a neutralization assay. Laboratory investigation
For this study, serum and CSF samples of patients with potential WNV infections were tested for the presence of
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specific IgM and IgG antibodies (WNV IgM Capture DxSelect and WNV IgG DxSelect by Focus Diagnostics, Cypress, USA). Furthermore, the presence of WNV-specific antibodies was confirmed by a plaque-reduction neutralization test (PRNT) (Barzon et al. 2013b). Serum, CSF, and urine samples, collected during the acute phase, were examined for the presence of WNV RNA with three methods— a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) targeting a conserved region of WNV lineages 1 and 2 (Linke et al. 2007) and two pan-Flavivirus nested RTPCRs (Scaramozzino et al. 2001, Sa´nchez-Seco et al. 2005) followed by sequencing of amplicons. In addition, WNV RNA was searched for in urine samples as previously suggested (Barzon et al. 2013b). Furthermore, 455 stored CSF samples collected in six previous spring–autumn seasons, 2007–2012, which had been previously scored as negative for bacterial and virus infections, were investigated retrospectively to evaluate the potential unreported presence of WNV infection in Lombardy. Entomological and veterinary surveillance of WNV infections
From July to October, 2013, national and regional institutions implemented the entomological and veterinary surveillance program related to WNV in the Lombardy Region. The entomological surveillance is based on a range of collection sites placed either in at-risk areas or in surrounding areas and aims at identifying possible WNV vector species as well as at determining their abundance and distribution. Entomological sampling was performed in four sites from the beginning of the season, whereas 23 sites were additionally activated from the beginning of September in the frame of a regional surveillance plan. Mosquitoes were collected by attractive traps, grouped in pools according to species, data, and sites of sampling, and screened by molecular tests for the presence of WNV (Calzolari et al. 2013). A total of more than 20,000 mosquitoes were collected. The veterinary surveillance system was based upon testing of sentinel birds and horses with clinical signs of potential WNV infection. In the period July–October of 2013, 632 wild birds were sampled and tested by a WNV-specific molecular test (Tang et al. 2006). Ethics statement
The local Ethics Committee consent was not required because according to a Regional Surveillance and Preparedness Plan (DGR 12591, December 27, 2012), diagnostic detection of WNV infections in the Lombardy Region was centralized at the Regional Reference Laboratory (Molecular Virology Unit, Fondazione IRCCS Policlinico San Matteo, Pavia). Informed consent was not necessary because patients with suspected WNV infections were included in a Regional diagnostic protocol. Prospective samples (serum, CSF, and urine) were collected by clinicians and handled by Molecular Virology Unit personnel; data were analyzed anonymously according to a Regional Surveillance and Preparedness Plan (DGR 12591, December 27, 2012). The 455 control CSF samples were collected and stored during the period 2007– 2012 from as many hospitalized patients with neurologic disorders as surplus volume following completion of routine diagnostic assays. All samples had been previously scored as
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negative for bacterial and virus infections and were investigated retrospectively to evaluate the potential unreported presence of WNV infection in Lombardy. This retrospective analysis was performed according to guidelines of the Institutional Review Board of the Fondazione IRCCS Policlinico San Matteo on the use of biologic specimens for scientific purpose in keeping with Italian law (art.13 D.Lgs 196/2003). The personnel of Molecular Virology Unit participated in the collection of these samples, which were anonymized upon collection. Results
As shown in Table 1, in the period from August 13 to October 4, 2013, 10 confirmed cases of acute WN-ND and eight cases of acute WNF (six confirmed and two probable) were diagnosed. In Figure 1A, the weekly distribution of WNV cases is shown. WNV cases were distributed in four different provinces—[]]Cremona (n = 9), Mantua (n = 5), Brescia (n = 3), and Lodi (n = 1)—in the southeastern area of the Lombardy Region, in close proximity to areas of the Emilia-Romagna and Veneto Regions affected by WNV outbreaks in previous years (Fig. 1B). Among patients with WN-ND, nine (90%) were males and one (10%) female, with a median age of 75 years (range, 54– 89). WN-ND cases were detected in all the four provinces involved in the WNV outbreak (Table 1). All WN-ND patients required hospitalization: Eight presented with encephalitis, three of them died, and two presented with meningoencephalitis. WNV-specific IgM antibodies were detected in all serum samples and in seven CSF specimens.
WNV-specific IgG antibodies were detected in 10 serum samples and in four CSF. In nine WN-ND cases, the specificity of the WNV antibodies was confirmed by the neutralization test, whereas in one case the assay was not performed (Table 1). WNV RNA was detected in the serum sample of one patient and in the CSF and serum of another patient. WNV viruria was observed in a WN-ND patient, in the absence of CSF and serum positivity for WNV RNA (Table 1). Of the eight patients with WNF, four (50%) were males and four (50%) females, with a median age of 58 years (range, 17–87). The cases were mostly detected in patients living in the province of Cremona (n = 7) and one in the province of Mantua. Six were confirmed cases of WNF and two were probable. The most common clinical signs were fever (n = 8) and rash (n = 2). WNV-specific IgM antibodies were detected in all eight serum samples, whereas WNVspecific IgG antibodies were detected in six serum samples. The specificity of the WNV antibodies was confirmed by the neutralization test in six cases, whereas in two cases the assay was not performed (Table 1). WNV RNA was detected in the urine of a single patient (Table 1). Sequencing of the NS5 gene showed that all WNV strains belonged to lineage 2
Evidence of this outbreak during the summer of 2013 is further strengthened by the finding that among the 455 CSF samples collected during the six previous summer seasons (2007–2012) and tested for the presence of specific IgM and IgG antibodies and WNV RNA; only one was scored as IgG positive.
Table 1. Characteristics of Human WNV Infections, in the Lombardy Region, August–October, 2013 Age/ Patient sex 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 a
78/M 66/M 89/M 61/M 79/M 49/M 55/F 75/F 54/M 17/M 71/F 63/M 27/F 87/F 54/M 78/M 76/M 57/M
Origin
Clinical presentation
Mantua Mantua Mantua Mantua Mantua Cremona Cremona Cremona Cremona Cremona Cremona Cremona Cremona Cremona Brescia Brescia Brescia Lodi
Encephalitis Meningoencephalitis Encephalitis West Nile fever Encephalitis West Nile fever West Nile fever Encephalitis Encephalitis West Nile fever West Nile fever West Nile fever West Nile fever West Nile fever Encephalitis Encephalitis Meningoencephalitis Encephalitis
ELISA IgMa ELISA IgGb
RT-PCRc,d
Outcome Serum CSF Serum CSF Neutralization Serum CSF Urine Alive Alive Dead Alive Dead Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Dead
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1*f 1 1 1
1 1 1 NA 1 NA NA 1 1 NA NA NA NA NA -f 1 NA NA
1 1 1 1 1 1 1 1 1 1 1 1 1*f 1 1 1
1 NA 1 NA NA 1 NA NA NA NA NA -f 1 NA NA
1 1 1 ND 1 1 ND 1 1 1 1 1 1 1 ND 1 1 1
1 -e -
2 NA NA NA NA NA NA NA NA +e NA NA
NA NA NA 1 NA NA NA NA 1 +e NA -
WNV IgM Capture DxSelect (Focus Diagnostics). WNV IgG DxSelect (Focus Diagnostics). c Real-time RT-PCR WNV L1-2 (Linke et al 2007). d Nested RT-PCR pan-Flavivirus (Scaramozzino et al. 2001, Sa´nchez-Seco et al. 2005). e Real-time RT-PCR Flavivirus (Moureau et al. 2007). f WNV IgG/IgM IIFT (Euroimmun); *Convalescent serum sample. N WNV, West Nile virus; ELISA, enzyme-linked immunosorbent assay; IgM, immunoglobulin M; CSF, cerebrospinal fluid; M, male; F, female; NA, not available; ND, not done. b
WEST NILE OUTBREAK IN LOMBARDY
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FIG. 1. (A) Human cases of West Nile virus (WNV) infection by week of symptom onset, Lombardy Region, August– October, 2013. (B) Distribution of human and animal cases of WNV infection, Lombardy Region, summer, 2013. WNND, WNV neuroinvasive disease. Color images available online at www.liebertpub.com/vbz
The veterinary and entomological surveillance programs to investigate WNV in the Lombardy Region revealed the presence of six WNV positive pools of Culex pipiens, the main vector of WNV in Italy, in the provinces of Cremona (n = 2), Mantua (n = 2), and Brescia (n = 2) (Fig. 1B, Tables 2 and 3). Eleven infected horses were detected in province of Mantua (n = 3) and Cremona (n = 8) (Fig. 1B, Table 2). An
infected crow was detected in the province of Cremona (Fig. 1B, Table 2). Discussion and Conclusion
WNV was detected for the first time in the Lombardy Region in September, 2009, with two confirmed cases of
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Table 2. WNV Positivity in the Provinces of Lombardy Region during Veterinary Surveillance in 2013 Province
Cases
Date
Species
Mantua
3
September 02 August 28 August 06 September 23 August 09 August 25 September 18 October 07 August 08
Horses (seroconversion) Insects
2 Cremona
4
4 2 Brescia
1 2
fection prior to 2013 in Lombardy (http://sorveglianza.izs.it/ emergenze/west_nile/emergenze.html). As previously shown (Calzolari et al. 2013), the veterinary and entomological surveillance plans allow for detecting WNV circulation much earlier than the occurrence of the human cases and provide important epidemiological information on the circulating strains in a certain area that could infect humans. Having such a rapid health alarm system in place is of extreme value because it enables regional infrastructures for optimal management of acute human cases and adoption of prevention strategies. Sequencing of NS5 amplicons in two human cases of WNND showed > 95% nucleotide identity with WNV Rovigo strains (lineage 2) circulating in the Veneto Region in 2013 (GenBank acc. nos. KF647251.1 and KF5883365.1). Finally, our data support the diagnostic value of WNV detection in urine samples of infected patients (Barzon et al. 2013b) and stress the importance of its routine use. In conclusion, an outbreak of WNV in the summer of 2013 involving the southeastern provinces of the Lombardy Region is described as a part of the larger outbreak involving the neighboring Emilia-Romagna and Veneto Regions. This is the largest outbreak of WNV reported in Lombardy so far, showing a west bound spread of the virus after 5 years spread in a northeastern direction.
Horses (with clinical signs) Horses (seroconversion) Insects
July 04 September 06 August 29 September 05 September 19
Crow Insects
WN-ND in the province of Mantua (Rizzo et al. 2012). A seroprevalence study to detect anti-WNV–specific antibodies was carried out in blood donations in the metropolitan area of Milan in the summers of 2009 and 2011; no positive sera were found in 2009, whereas five positive sera were detected in 2011 (Gaibani et al. 2013). These data, along with an IgGpositive CSF sample in our retrospective series, would suggest the presence of individuals with subclinical WNV infection in Lombardy (Gaibani et al. 2013). However, the area where the infection was acquired could not be determined and it cannot be excluded that it was acquired when traveling in surrounding regions affected by the epidemics since 2008. On the other hand, the substantial absence of WNV-positive CSF over a period of 6 years would suggest that in Lombardy the virus was not actively circulating before the summer of 2013. The veterinary and entomological surveillance programs for WNV confirmed the presence of WNV infection in mosquitoes, horses, and birds in Lombardy in summer of 2013, further supporting the timing of the WNV outbreak in this region. Indeed, the surveillance programs conducted in horses and wild birds since 2009 confirm the absence of in-
Acknowledgments
We thank all of the technical staff for handling the specimens and performing the assays. We thank Daniela Sartori for manuscript editing and Laurence Kelly for English language revision. This work was supported by a grant from the Regional Health Authority of Lombardy, Milan, Italy (DGR no. 12591 of December 27, 2012). Members of the West Nile Virus Task Force are as follows: Virology Units—city and participants: Pavia (Fausto Baldanti, Francesca Rovida, Antonella Sarasini, Giulia Campanini, Elena Percivalle, Giovanna Gorini, Bianca Mariani); Roma (Maria Rosaria Capobianchi, Concetta Castilletti). Infectious and Tropical Diseases Unit—city and participants: Cremona (Angelo Pan, Fabio Zacchi); Brescia (Francesco Castelli). Infectious Diseases Unit—city and participants: Mantova (Paolo Costa, Lisa Manzini); Varese (Paolo Antonio Grossi).
Table 3. Mosquitoes Collected in Lombardy Region During Entomological Surveillance in (July–October, 2013) Brescia Species Aedes (Ae.) albopictus Ae. caspius Ae. cinereus Ae. geniculatus Ae. vexans Aedes spp. Anopheles maculipennis Culiseta annulata Culex (Cx.) modestus Cx. pipiens Total
n
Cremona
Pools
n
Lodi
Mantua
Total
Pools
n
Pools
n
Pools
n
Pools
n
Pools
3 2475 12 3 2249 1 1940 6 30 6752 13,471
3 30 3 2 30 1 21 6 7 51 154
121 2744 12 3 2368 7 1959 9 52 13527 20,802
37 42 3 2 38 3 25 8 10 125 293
8 1
6 1
68
11
26 6
10 2
16 262
7 9
2 6
1 2
7
3
3
1
107
3
1
1
2 15 3988 4022
1 2 20 33
193 229
13 27
18 1 7 580 991
3 1 1 19 43
2014 2089
Pavia
22 36
WEST NILE OUTBREAK IN LOMBARDY
Emergency Unit—city and participants: Cremona (Antonio Cuzzoli). Nephrology and Dialysis Unit—city and participants: Cremona (Stefano Possenti). Transplantation Unit, Nephrology—city and participants: Brescia (Nicola Bossini). Veterinary Unit—city and participants: Brescia (Mattia Calzolari, Davide Lelli, Antonio Lavazza). Regional Health Authority of Lombardy (Maria Gramegna, Alessandra Piatti, Piero Frazzi). Author Disclosure Statement
No competing financial interests exist. References
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Address correspondence to: Fausto Baldanti Molecular Virology Unit Microbiology and Virology Department Fondazione IRCCS Policlinico San Matteo 27100 Pavia Italy E-mail: [email protected]
