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Two Clusters of Toscana Virus Meningo-Encephalitis in Livorno Province and Elba Island, July-September 2018

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Two Clusters of Toscana Virus Meningo-Encephalitis in Livorno Province and Elba Island, July-September 2018 Ann Ig 2020; 32(6): 674-681 doi:10.7416/ai.2020.2387 Two clusters of Toscana virus meningo-encephalitis in Livorno Province and Elba Island, July-September 2018 F. Quattrone1, P. Mazzetti2,3, F. Aquino4, S. Sani5, L. Carneglia4, M. Pistello2,3, P.L. Lopalco1, L. Tavoschi1 Key words: Vector-borne disease, Toscana virus, outbreak, Italy Parole chiave: Malattie trasmesse da artropodi, virus Toscana, focolaio epidemico, Italia Abstract Background. Toscana virus (TOSV) is an arbovirus transmitted to humans by Phlebotomus spp sandflies. It causes aseptic meningitis and meningoencephalitis with marked seasonality. Here we describe the clinical, microbiological and epidemiological features of two clusters of cases occurred in Tuscany in 2018. Methods. A confirmed case was defined as the detection of anti-TOSV IgM and IgG in serum sample, in presence of typical clinical manifestations. We consulted hospital records of hospitalized patients to collect clinical information and obtained epidemiological information from the local health authority investigation report. We telephonically interviewed patients using a standard questionnaire for a 6 months follow-up. Results. A total of 12 cases of TOSV meningo-encephalitis with onset between 4th of July and 12th of September accessed health care services in the province of Livorno. Eight cases were males with median age 41,5 and four were not resident in the area. Serological investigations confirmed a recent TOSV infection. Eight cases reported visiting Elba Island and four had a possible occupational-related exposure. Conclusions. This surge of infection emphasizes the need of information campaigns coupled with adequate surveillance and control interventions against TOSV that, among other arboviruses, is a growing issue of concern in Italy. Introduction Phlebotomus perniciosus (1). The virus can be transmitted to humans through the Toscana virus (TOSV) is an arbovirus bite of an infected female sandfly and was belonging to the sandfly fever Naples associated with human disease in early complex of the Phenuiviridae family, 1980s (2). Although infections are often first identified in 1971 from the sandfly asymptomatic, TOSV was reported as one 1 Hygiene and Epidemiology Section, Department of Translational Research and of New Surgical and Medical Technologies, University of Pisa, Pisa, Italy 2 Virology Unit, Azienda Ospedaliero-Universitaria Pisana, Pisa, Italy 3 Retrovirus Center and Virology section, Department of Translational Research and of New Surgical and Medical Techno- logies, University of Pisa, Pisa, Italy 4 Public Health Unit, ASL Toscana Nord Ovest, Pisa, Italy 5 Infectious Diseases Unit, Livorno Hospital, Livorno, Italy Two clusters of Toscana virus meningo-encephalitis 675 of the most important etiological agents Time Reverse Transcriptase-Polymerase of viral meningitis in Italy (3, 4) and other Chain Reaction (RT-PCR) Kit (Liferiver, Mediterranean countries (5, 6). The disease, Shanghai, China). In brief, Viral RNA was prevalent among adults, has a marked extracted from 200 ml of serum e/o liquor seasonality with cases most often occurring using Qiasymphony instrument (Qiagen) during late summer (3, 4). The ecology of the and a 5 ml RNA sample was used for the virus is not well known, although antibodies Real-time reaction. Anti-TOSV antibodies against the virus have been detected in were investigated using commercial enzyme- various vertebrates including horses, sheep linked immunosorbent assay (ELISA) tests and dogs (4, 7). Circulation of the virus in for TOSV IgG and IgM specific antibodies Tuscany and its islands is well described in (Enzywell Toscana Virus – DIESSE Siena, the literature and seroprevalence surveys of Italy) and anti-West Nile antibody using the resident population reported up to 30% SERION ELISA classic West Nile Virus positivity to anti-TOSV antibodies (4, 8). IgG/IgM (Institut Virion\Serion GmbH, Besides, Tuscany is a world-famous Wurzburg, Germany). holiday destination and cases in returning West-Nile and Chikungunya Virus travelers have also been described over the RNA genome search was performed, years (9, 10) and also more recently (11). respectively, with WNV ELITe MGB Kit Yet, current surveillance activities in the (Elitech, Puteaux, France) and Chikungunya region and at national level are suboptimal. Virus Real Time RT-PCR Kit (Liferiver, Here we describe two clusters of TOSV Shanghai, China) and Usutu genome cases occurred in Tuscany during July- search with a home-made Real-time RT- September 2018, in order to increase PCR (13, 14). Confirmatory test was awareness about this arbovirosis (12). performed with ELISA Test (as above) and virus neutralization. Virus neutralization was performed on Vero cell monolayers Materials and methods by plaque reduction neutralization tests (PRNT). Neutralization antibody titres were A confirmed case was defined as the calculated as the reciprocal of the serum detection of anti-TOSV IgM and IgG in dilution that gave an 80% reduction of the serum sample, in presence of typical clinical number of plaques (NT80), as compared to manifestations. the virus control. Titres ≥10 were considered All virological and serological analyses positive and all the 7 sera sent to the ISS had were performed on plasma samples by the a title PRNT80 ≥1:10. Virology Unit of the Pisa University Hospital, All patients corresponding to the case Regional Reference Center for Arboviruses definition during the period July-September and the only center performing TOSV 2018 in the database of the Virology Unit of serology in the Toscana North-West Local Pisa were included in the study. Health Authority (LHA) to which Livorno Hospital records of hospitalized patients and Elba island refer. The samples were in Livorno hospital (referral center for collected from symptomatic patients by the Infectious disease of Leghorn Province Livorno Infectious Disease Unit and sent to and Elba Island) were consulted to collect the Pisa reference center. Positive cases were clinical information. In particular, data were confirmed by the Italian National Institute of collected in an anonymous form using a Health (Istituto Superiore di Sanità, ISS). standard set of variables. The variables Search for TOSV RNA genome was extracted were sex, age, clinical diagnosis, performed using the Toscana Virus Real date of beginning of symptoms, use of 676 F. Quattrone et al. Emergency Departments (ED) services, Eight of the twelve cases were males, use of intensive care unit, characteristics aged 22-51 years (median 41.5); eight were of symptoms, characteristics of liquor, resident in Tuscany (Table 1). date of hospitalization, number of days of All patients scored positive for anti- hospitalization, health status at discharge, TOSV IgM and IgG in serum samples. Five risk factors of the subject. Epidemiological patients were tested for the presence of the information was obtained from the LHA viral genome and scored negative (Table investigation report. Telephonic interviews 1). All patients scored negative for West with patients were performed in February- Nile virus antibodies, one and two patients March 2019 using a standard questionnaire were also negative for Usutu virus and to make a 6-month follow-up and collect Chikungunya virus antibodies, respectively. additional information. Serum samples obtained from the seven patients belonging to the second cluster underwent confirmatory testing and scored Results positive both with ELISA and neutralization assays. In September 2018 a cluster of seven As for the interviews, one patient refused cases of TOSV meningo-encephalitis, to participate, and one could not be contacted. with onset between 30th of August and 12th Respondents reported having been, for of September, was reported to the LHA work or leisure, in the south-eastern part in Livorno. A retrospective analysis of of Elba Island or in rural areas of South laboratory records led to the identification of Tuscany (Province of Livorno) (Figure 2). a previous cluster of five patients with onset In particular, 8 of the 11 cases for whom between 4th and 12th July in the same area the information was available had been on (Figure 1). The LHA launched an outbreak the Elba Island during the 15 days before investigation in September 2018. symptoms onset. The most common reported Figure 1 - Confirmed cases of TOSV meningo-encephalitis, Tuscany, Italy, 4 July -12 September 2018 Two clusters of Toscana virus meningo-encephalitis Table 1 - Demographic, clinical, and epidemiological characteristics of cases Age Onset Molecular diagnosis Healthcare Case Sex Clinical presentation Risk factors (y) (Week) and serology(Blood) services Aseptic meningitis, asthenia, low fever, TOSV IgM+ TOSV IgG + 2 ER accesses Resident in Livorno district, hiking 1 M 33 27 headache, nausea and vomiting, photopho- TOSV genome – 7d Hospital stay in naturalistic area bia, arthralgia Aseptic meningitis, asthenia, high fever, TOSV IgM+ TOSV IgG + 1 ER access Resident in Livorno, gardening, 2 F 43 28 headache, photophobia phonophobia, arth- TOSV genome – 6d Hospital stay beach attendance ralgia, rigor nucalis, vertigo Aseptic meningitis, asthenia, drowsiness, Resident on Elba Island, beach at- TOSV IgM+ TOSV IgG + 1 ER access 3 F 45 28 low fever, headache, photophobia arthralgia, tendance, self-reported exposure to TOSV genome – 5d Hospital stay rigor nucalis, vertigo sandfly bites Aseptic meningitis, asthenia, high fever, 2 ER access 4 M 46 28 TOSV IgM+ TOSV IgG + headache, nausea and vomiting, vertigo,
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