Antonella Marchi

Encephalitis without Meningitis Due to Sandfly Fever Virus Serotype Toscana

The role of Toscana (TOS) virus in producing encephalitis without meningitis is uncertain. We studied 2 cases of TOS virus encephalitis without meningitis by means of nested polymerase chain reaction assay and DNA sequencing. Findings confirm that TOS virus may directly cause encephalitis and suggest the usefulness of DNA sequencing in investigating relationships between TOS virus molecular patterns and the spectrum of neurological involvement.

Unusual Presentation of Life-Threatening Toscana Virus Meningoencephalitis

This case report describes a brother and a sister with severe meningoencephalitis caused by Toscana virus (TOSv). The clinical presentation was characterized by stiff neck, deep coma, maculopapular rash, diffuse lymphadenopathy, hepatosplenomegaly, renal involvement, tendency to bleeding, and diffuse intravascular coagulation. The boy had epididymo-orchitis. Recovery with neurologic sequelae as hydrocephalus was observed. Microbiological diagnosis was obtained by serological tests and reverse transcriptase-polymerase chain reaction. Sequencing of polymerase chain reaction products from the S and M segments was carried out. TOSv may be a causative agent in severe meningoencephalitis.

Circulation of West Nile virus lineage 1 and 2 during an outbreak in Italy.

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.

Viral Isolates of a Novel Putative Phlebovirus in the Marche Region of Italy

Thirty pools from 900 (540 females and 360 males) Phlebotomus perfiliewi sandflies collected during the summer of 2012 in the Fermo area (Marche Region, central Italy) were tested for the presence of Phleboviruses. A nested polymerase chain reaction was performed using degenerated primers amplifying a fragment of the polymerase gene (large segment) and a fragment of the nucleoprotein gene (small segment) of the genus Phlebovirus. One pool was positive for Toscana virus, as expected from results of studies in the area, and six pools were positive for a putative novel Phlebovirus. Virus isolation in Vero cells was performed. Minimum field infection rates/1,000 insects processed for the novel and Toscana viruses were 6.7 and 1.0, respectively. Phylogenetic analysis of the novel Phlebovirus, tentatively named Fermo virus, placed it in the Sandfly Fever Naples virus serocomplex.

Chikungunya and Dengue Viruses in Travelers

To the Editor: Chikungunya virus (CHIKV), an arthropod-borne virus transmitted to humans by Aedes spp. mosquitoes, was first isolated in Tanzania (Tanganyika) in 1953 (1). Various outbreaks have since occurred in Africa, Southeast Asia, and India (2). CHIKV has recently been reported in a large area in the Indian Ocean islands and the Indian subcontinent. After an outbreak in Kenya in 2004, other outbreaks occurred in early 2005 on the Comoros Islands, Reunion, and other islands in the southwestern Indian Ocean; the epidemic then spread to India (3,4). Molecular analysis showed that the epidemic was caused by a variant of the Central/East African CHIKV genotype (5,6). Internet surveillance networks provided information on epidemics in real time, alerting clinicians in the industrialized world to the spread of CHIKV and enabling them to more easily diagnose infection among travelers with fevers (7). We report results of diagnostic tests and analysis of predictors of infection among persons in Italy with symptoms suggestive of CHIKV infection who had traveled to potentially affected areas. Dengue virus (DENV) is endemic to many of these areas. We studied travelers or migrants from areas to which CHIKV infection is endemic (i.e., sub-Saharan Africa) or areas currently affected by outbreaks (i.e., the Indian Ocean islands, India) who had symptoms suggestive of infection (i.e., fever and arthralgia with or without a rash) from January 2006 through March 2007. At least 1 blood sample was collected from each patient and stored at –80°C before testing for CHIKV and DENV. Median lag between onset of symptoms and date of blood collection was 22 days (range 3–179 days). Two samples (acute phase and convalescence phase) were available from 5 patients. Serologic diagnosis of CHIKV infection was determined by hemagglutination inhibition (HI) test and confirmed by plaque-reduction neutralization test (8). Serodiagnosis of DENV infection was conducted by using the HI test and an immunoglobulin M ELISA (Focus Diagnostics, Cypress, CA, USA). A case-report form containing information about age, sex, countries visited, travel dates, and date of onset of symptoms was completed for each patient. Seventy-six persons participated in the study; 55.3% were male, median age was 39 years (range 1–69 years), and most (80.3%) were Italian (Table). A total of 29 (38.2%) were positive for CHIKV, and 13 (17.1%) were positive for DENV; 34 (44.7%) were negative for both viruses. Of the 29 CHIKV-positive persons, 22 (75.9%) had visited the Indian Ocean islands (Mauritius, Reunion, and Madagascar), 5 had visited Asia, and 2 had visited Africa. Travelers from Indian Ocean islands had a higher risk for CHIKV infection than those who had visited Africa (odds ratio [OR] 11.0, 95% confidence interval [CI] 1.60–119.13) or Asia (OR 17.05, 95% CI 4.31–73.05). Persons who had visited Asia had a higher risk for DENV infection (OR 8.36; 95% CI 1.58–81.73) than those who had visited other areas. Table Characteristics of 76 travelers studied The 5 persons who were infected with CHIKV in Asia had visited India (i.e., the most visited country [21 travelers]). However, persons who visited the Indian Ocean islands had a higher risk of being CHIKV positive than those who had visited India (OR 8.8, 95% CI 2.09–39.86). A rash was associated with CHIKV infection and was >8× more likely to be reported by CHIKV-positive persons than CHIKV-negative persons (OR 7.03, 95% CI 2.23–22.93). Moreover, rash was observed in 65% of CHIKV-positive cases and 31% of DENV-positive cases, but the difference was not statistically significant because of the small sample size (OR 4.28, 95% CI 0.88–23.23). None of the other patient’s characteristics was associated with infection with CHIKV or DENV. A limitation of our study was that only 5 patients had documented seroconversion for CHIKV. However, high titers were found in all but 1 patient (>1,280 in 21 patients and 640 in 2 patients). This patient, who had a titer of 80, was an Italian who had probably not been previously exposed to CHIKV. Thus, the risk for misclassification was low. PCR for early detection of infection was not used because only 3 persons were tested within 10 days of symptom onset. Two of these persons, who were tested 7 days after symptom onset, already had antibodies to CHIKV. In conclusion, a high proportion of travelers with symptoms of CHIKV infection who returned from areas with outbreaks of this infection or where this virus was endemic were seropositive. A lower proportion of patients had antibodies to DENV. CHIKV-positive patients were more likely to have a rash than those negative for both CHIKV and DENV. As suggested by previous studies (9), a rash was more common among CHIKV-positive patients than in DENV-infected patients, but the difference was not significant. Our study suggests that identification of predictors of infection with CHIKV is feasible, although it is complicated by cocirculation of DENV in the same areas.

Humoral immunity in natural infection by tick-borne encephalitis virus.

Tick‐borne encephalitis (TBE) virus is one of the most important flaviviruses associated with neurological disease in Europe. Cross‐reactive antibodies elicited by different flaviviruses can make difficult the interpretation of ELISA and hemagglutination‐inhibition (HI) tests for the diagnosis of TBE. Neutralization tests, which are more specific, are not in common use because they are difficult to perform and standardize. A plaque reduction neutralization test (PRNT), optimized previously in vaccinated children, was evaluated in sera from acute cases of TBE, collected for diagnostic purposes, and from healthy human population and wild ruminants, collected for serosurvey purposes. The PRNT results were compared with the results of ELISA and HI tests. In acute TBE disease, most sera were positive for IgM antibodies by ELISA and with high HI antibody titers; neutralizing antibodies were detected in 71.4% of patients, at a very low titer (1:10 NT50) in almost all cases. Seroprevalences of 8% and 6.5% for anti‐TBE ELISA antibodies were found in healthy subjects and wild ruminants, respectively. Among anti‐TBE positive healthy subjects, a very low 1:10 NT50 titer was detected in 17.4% of cases, while NT80 titers ranging from 1:10 to 1:80 were detected in 65.2% of cases. Among wild ruminants, 90.9% of ELISA and HI positive samples showed a positive, ≥1:10 NT80 titer. In conclusion, neutralization assays can be useful for the diagnosis and serosurveys of TBE. J. Med. Virol. 81:665–671, 2009

Measles elimination in Italy: data from laboratory activity, 2011–2013

BACKGROUND The European Regional Office of the World Health Organization developed a strategic approach to halt the indigenous transmission of measles in its 53 Member States by 2015, World Health Organization [1]. Many European countries, including Italy began the implementation of national programs to reach this goal. OBJECTIVES To describe and discuss the results of laboratory activity in measles surveillance, performed from January 2011 to December 2013 by the Italian National Reference Laboratory for Measles and Rubella. STUDY DESIGN Samples of suspected measles cases were collected from different Italian regions to confirm clinical diagnosis. Anti-measles IgM antibodies detection by Enzyme-Linked Immunosorbent Assay and/or molecular detection by Reverse Transcriptase-Polymerase Chain Reaction assay were performed. Positive samples were sequenced for viral characterization. RESULTS AND CONCLUSIONS According to results from the National Reference Laboratorys activity urine and blood seem to be the best specimens for measles laboratory surveillance. Phylogenetic analysis revealed a co-circulation of the genotypes D4 and D8 during the reviewed period, a cluster of B3 and sporadic cases of D9 and H1.

Serological evidence of Toscana virus infection in Portuguese patients

Toscana virus (TOSV) is an emerging Phlebovirus of growing interest as a human pathogen in the Mediterranean Basin. In Portugal, however, little is known about the prevalence of TOSV infection. The aim of this work was to perform a seroprevalence study in patients with requests for laboratory diagnosis of vector-borne viruses. A total of 538 patients with and without neurological signs from 2004 to 2008 were studied by in-house indirect immunofluorescence assay and commercial enzyme-linked immunosorbent assays. A prevalence of 4.2% for IgG antibodies was found in the group of patients with neurological signs. Five (3%) of these had recent infections. In the group with no neurological signs, the IgG prevalence was 1.3%. Two samples, belonging to two patients, were also confirmed with plaque reduction neutralization tests with the TOSV ISS. Phl.3 Italian strain. This work showed that TOSV is present and causing disease from north to south in Portugal. The probable circulation of different phlebovirus serotypes in Portugal emphasizes the need for further studies.

Prevalence of antibodies to phleboviruses and flaviviruses in Peja, Kosovo

In order to investigate the current and past activity of phlebovirus and flavivirus in Kosovo, a seroprevalence study among 200 blood donors was performed. Positive results were obtained for the phleboviruses TOSV and SFNV, and for a flavivirus of the Japanese Encephalitis group. No positive results for TBEV were observed.

Molecular epidemiology of measles virus in Italy, 2002–2007

BackgroundThe European Regional Office of the World Health Organization (WHO/Europe) developed a strategic approach to halt the indigenous transmission of measles in its 53 Member States by 2015. In view of the goal of measles elimination, it is of great importance to assess the circulation of wild-type measles virus (MV). Genetic analysis is indispensable to understand the epidemiology of measles.MethodsUrine and saliva samples were collected between May 2002 and December 2007, in order to find the origins and routes of wild type measles virus circulation. RT-PCR was performed on a total of 414 clinical samples of patients from different Italian regions. The results confirmed the genome presence in 199 samples, out of which 179 were sequenced. The sequences were genotyped by comparing the fragment coding for the carboxyl terminus of the nucleoprotein (450 nucleotides) with that one of the WHO reference strains.ResultsFrom the year 2002 to the year 2007 phylogenetic analysis of measles sequences showed a predominant circulation of the D7 genotype in the Italian territory for the years 2002–2004. This genotype was replaced by D4 and B3 genotypes in the biennium 2006–2007. During the same period C2, A, D5 and D8 genotypes were also detected.ConclusionsGenetic characterization of wild-type MV provides a means to study the transmission pathways of the virus, and is an essential component of laboratory-based surveillance. Knowledge of currently circulating measles virus genotype in Italy will help in monitoring the success of the measles elimination programme and will contribute to evaluate the effectiveness of future vaccination campaigns.