Clinical features and prognostic factors in adults with viral meningitis.

Clinical features applicable to the entire spectrum of viral meningitis are limited, and prognostic factors for adverse outcomes are undetermined. This nationwide population-based prospective cohort study included all adults with presumed and microbiologically confirmed viral meningitis in Denmark from 2015 until 2020. Prognostic factors for an unfavourable outcome (Glasgow Outcome Scale score of 1-4) 30 days after discharge were examined by modified Poisson regression. In total, 1066 episodes of viral meningitis were included, yielding a mean annual incidence of 4.7 episodes per 100 000 persons. Pathogens were enteroviruses in 419/1066 (39%), herpes simplex virus type 2 in 171/1066 (16%), varicella-zoster virus in 162/1066 (15%), miscellaneous viruses in 31/1066 (3%) and remained unidentified in 283/1066 (27%). The median age was 33 years (IQR 27-44), and 576/1066 (54%) were females. In herpes simplex virus type 2 meningitis, 131/171 (77%) were females. Immunosuppression [32/162 (20%)] and shingles [90/149 (60%)] were frequent in varicella-zoster virus meningitis. The triad of headache, neck stiffness and hyperacusis or photophobia was present in 264/960 (28%). The median time until lumbar puncture was 3.0 h (IQR 1.3-7.1), and the median CSF leucocyte count was 160 cells/µl (IQR 60-358). The outcome was unfavourable in 216/1055 (20%) 30 days after discharge. Using unidentified pathogen as the reference, the adjusted relative risk of an unfavourable outcome was 1.34 (95% CI 0.95-1.88) for enteroviruses, 1.55 (95% CI 1.00-2.41) for herpes simplex virus type 2, 1.51 (95% CI 0.98-2.33) for varicella-zoster virus and 1.37 (95% CI 0.61-3.05) for miscellaneous viruses. The adjusted relative risk of an unfavourable outcome was 1.34 (95% CI 1.03-1.75) for females. Timing of acyclovir or valacyclovir was not associated with the outcome in meningitis caused by herpes simplex virus type 2 or varicella-zoster virus. In summary, the outcome of viral meningitis was similar among patients with different aetiologies, including those with presumed viral meningitis but without an identified pathogen. Females had an increased risk of an unfavourable outcome. Early antiviral treatment was not associated with an improved outcome in meningitis caused by herpes simplex virus type 2 or varicella-zoster virus.

Untargeted metagenomic sequencing identifies Toscana virus in patients with idiopathic meningitis, southern Spain, 2015 to 2019.

BackgroundVarious pathogens, including bacteria, fungi, parasites, and viruses can lead to meningitis. Among viruses causing meningitis, Toscana virus (TOSV), a phlebovirus, is transmitted through sandfly bites. TOSV infection may be suspected if patients with enterovirus- and herpesvirus-negative aseptic (non-bacterial) meningitis recall recent insect bites. Other epidemiological factors (season, rural area) may be considered. The broad range of possible meningitis aetiologies poses considerable diagnosis challenges. Untargeted metagenomic next-generation sequencing (mNGS) can potentially identify pathogens, which are not considered or detected in routine diagnostic panels.AimIn this retrospective, single-centre observational study, we investigated mNGS usefulness to understand the cause of meningitis when conventional approaches fail.MethodsCerebrospinal fluid (CSF) samples from patients hospitalised in southern Spain in 2015-2019 with aseptic meningitis and no aetiology found by conventional testing, were subjected to mNGS. Patients' demographic characteristics had been recorded and physicians had asked them about recent insect bites. Obtained viral genome sequences were phylogenetically analysed.ResultsAmong 23 idiopathic cases, TOSV was identified in eight (all male; median age: 39 years, range: 15-78 years). Five cases lived in an urban setting, three occurred in autumn and only one recalled insect bites. Phylogenetic analysis of TOSV segment sequences supported one intra-genotype reassortment event.ConclusionsOur study highlights the usefulness of mNGS for identifying viral pathogens directly in CSF. In southern Spain, TOSV should be considered regardless of recalling of insect bites or other epidemiological criteria. Detection of a disease-associated reassortant TOSV emphasises the importance of monitoring the spread and evolution of phleboviruses in Mediterranean countries.

Infectious Toscana Virus in Seminal Fluid of Young Man Returning from Elba Island, Italy.

We report detecting infectious Toscana virus in the seminal fluid of a 25-year-old man from Italy returning from Elba Island. The presence of infectious virus in human semen adds Toscana virus to the long list of viruses detected in this genital fluid and indicates a potential for sexual transmission.

Human seroprevalence of Toscana virus and Sicilian phlebovirus in the southwest of Portugal.

Toscana virus (TOSV) is emergent in the Mediterranean region and responsible for outbreaks of encephalitis or meningoencephalitis. Sicilian phlebovirus (SFSV) cause epidemics of febrile illness during the summer. The aim of this study was to evaluate the presence of antibodies against TOSV and SFSV in humans in the southwest of Portugal. Neutralizing antibodies to TOSV and SFSV were respectively detected in 5.3% and 4.3% out of 400 human sera tested highlighting the need to increase public health awareness regarding phleboviruses and to include them in the differential diagnosis in patients presenting with fever of short duration and neurological manifestations.

Seroprevalence against Toscana virus in Spain: The case of the autonomous community of Madrid.

BACKGROUND & OBJECTIVES: The Toscana virus (TOSV) is a neurotropic arbovirus that is transmitted through the bite of some Phlebotomus species. In 2009, the largest outbreak of leishmaniasis described so far in Europe, occurred in the Autonomous Community of Madrid, Spain, which was related to the population increase of P. perniciosus in this region. METHODS: A seroprevalence study was conducted to determine the circulation of TOSV among the population of this geographic area. A total of 516 sera were collected in two different stages: 2007 (before the leishmaniasis outbreak) and 2018-19 (representative of the current situation). In the sera, presence of IgG antibodies against TOSV was determined by commercial ELISA. RESULTS: The overall seroprevalence was 34.5%. The anti-TOSV IgG level was significantly higher in the samples collected in 2007 (41.5%) than 2018-19 (27.3%). INTERPRETATION & CONCLUSION: The results of this study show a very active TOSV circulation in the region that is greater than expected. The lower seroprevalence figures in 2018-19 may be related to the vector and environmental control measures that were put in place as a result of the leishmaniasis outbreak of 2009. This highlights the importance of such strategies to reduce the incidence of TOSV infection and other vector-borne diseases.

Emergence of Toscana Virus, Romania, 2017-2018.

We describe a series of severe neuroinvasive infections caused by Toscana virus, identified by real-time reverse transcription PCR testing, in 8 hospitalized patients in Bucharest, Romania, during the summer seasons of 2017 and 2018. Of 8 patients, 5 died. Sequencing showed that the circulating virus belonged to lineage A.

Incubation Period for Neuroinvasive Toscana Virus Infections.

Toscana virus (TOSV) is an emerging pathogen in the Mediterranean area and is neuroinvasive in its most severe form. Basic knowledge on TOSV biology is limited. We conducted a systematic review on travel-related infections to estimate the TOSV incubation period. We estimated the incubation period at 12.1 days.

Field surveys in Croatia and North Macedonia reveal two novel phleboviruses circulating in sandflies.

Sandfly-borne phleboviruses are distributed widely throughout the Mediterranean Basin, presenting a threat to public health in areas where they circulate. However, the true diversity and distribution of pathogenic and apathogenic sandfly-borne phleboviruses remains a key issue to be studied. In the Balkans, most published data rely on serology-based studies although virus isolation has occasionally been reported. Here, we report the discovery of two novel sandfly-borne phleboviruses, provisionally named Zaba virus (ZABAV) and Bregalaka virus (BREV), which were isolated in Croatia and North Macedonia, respectively. This constitutes the first isolation of phleboviruses in both countries. Genetic analysis based on complete coding sequences indicated that ZABAV and BREV are distinct from each other and belong to the genus Phlebovirus, family Phenuiviridae. Phylogenetic and amino acid modelling of viral polymerase shows that ZABAV and BREV are new members of the Salehabad phlebovirus species and the Adana phlebovirus species, respectively. Moreover, sequence-based vector identification suggests that ZABAV is mainly transmitted by Phlebotomus neglectus and BREV is mainly transmitted by Phlebotomus perfiliewi. BREV neutralizing antibodies were detected in 3.3% of human sera with rates up to 16.7% in certain districts, demonstrating that BREV frequently infects humans in North Macedonia. In vitro viral growth kinetics experiments demonstrated viral replication of both viruses in mammalian and mosquito cells. In vivo experimental studies in mice suggest that ZABAV and BREV exhibit characteristics making them possible human pathogens.

Toscana virus encephalitis in Southwest Germany: a retrospective study.

BACKGROUND: Toscana virus (TOSV) is an arthropod-borne virus transmitted by phlebotomine sandflies (Phlebotomus sp.) widespread throughout the Mediterranean having the potential to cause meningoencephalitis in humans. In Germany, the vectors of TOSV are introduced recently and become endemic especially in Southwestern Germany. As TOSV is not investigated regularly in patients with meningoencephalitis, cases of TOSV-neuroinvasive disease may remain mostly undetected. METHODS: We conducted a retrospective cohort study on patients with meningoencephalitis without identification of a causal pathogen from 2006 to 2016. Serologic assessment for anti-TOSV-IgG and IgM was performed on serum and CSF. Demographic, clinical and CSF data from TOSV-positive patients were compared to a cohort of patients with meningoencephalitis due to enterovirus. Informed consent was obtained from all included patients. RESULTS: We found 138 patients with meningoencephalitis without identified causal pathogen. From 98 of these patients CSF and serum was available for further testing. Additionally, we included 27 patients with meningoencephalitis due to enterovirus. We identified two patients with serological confirmed TOSV-neuroinvasive disease (TOSV-IgM and IgG positive, 2%) and two patients with possible TOSV-neuroinvasive disease (isolated TOSV-IgM positive, 2%). Overall, TOSV-neuroinvasive was detected in 4% of our cases with suspected viral meningoencephalitis. None of them had a history of recent travel to an endemic area. CONCLUSIONS: We found cases of TOSV-neuroinvasive disease in our German cohort of patients with meningoencephalitis. As no recent history of travel to an endemic area was reported, it remains probable that these cases resemble autochthonous infections, albeit we cannot draw conclusions regarding the origin of the respective vectors. TOSV could be considered in patients with meningoencephalitis in Germany.

The initial detection of Toscana virus in phlebotomine sandflies from Turkey.

Toscana virus (TOSV) is a prominent arthropod-borne viral agent of human central nervous system infections occurring in the Mediterranean region. The main transmission route to susceptible individuals involves sandflies as vectors. Despite several reports revealing widespread TOSV activity in Turkey, vectors remained unidentified. A sandfly field survey was carried out in five provinces in Central, Southeast and Mediterranean Anatolia in 2017 to identify TOSV and related sandfly-borne phleboviruses and Leishmania parasites, with evidence for circulation in the region. A total of 7136 sandfly specimens, collected via standard methods, were evaluated in 163 pools. TOSV was detected in 11 pools (6.7%), comprising Phlebotomus major sensu lato, Sergentomyia dentata and Phlebotomus papatasi species. TOSV partial L and S segment sequences were characterized, that phylogenetically clustered with local and global genotype A strains. An amino acid substitution outside the conserved motifs of the viral polymerase, also present in previous TOSV sequences in endemic regions, was observed. Leishmania tropica was detected in a single pool of Ph. sergentii (0.6%). This is the first report of TOSV in sandflies from Turkey, and this study further provides evidence for additional sandfly species with the potential to transmit TOSV.

Acute cerebellar ataxia: a rare Toscana Virus (TOSV) meningoencephalitis complication.

Purpose: Arbovirosis, viral infection transmitted by arthropods, is a widespread health problem. In Italy, as well for all Mediterranean basin, from late spring to the end of summer, Toscana Virus (TOSV), a sandfly borne virus, accounts for the majority of aseptic meningitis/meningoencephalitis cases. TOSV meningitis/meningoencephalitis has usually a self-extinguishing benign course. Our aim is to report a case of a young healthy women diagnosed with Toscana Virus meningoencephalitis with a complicated clinical course.Materials and methods/results: Case report of a 33-years old woman, admitted to the Infectious Diseases Unit at Careggi General Hospital (Florence-Italy), with a diagnosis of Toscana Virus meningoencephalitis. Seventy-two hours after the admission, she developed typical symptoms, as impaired legs coordination, slurred speech, stumbling and dysmetria, of acute cerebellar ataxia (ACA). Urgent neurological assessment was provided performing an electroencephalography study followed by a brain and brainstem magnetic resonance imaging. In the meanwhile, bilateral nystagmus arised. Through neurologist consultation ACA clinical diagnosis was then made and intravenous steroid therapy was administered with prompt symptoms resolution. The patient was finally discharged at day 10 since the ACA onset in good clinical conditions.Conclusions: To raise awareness among physicians about possible neurological complications during Toscana Virus meningoencephalitis.

Two clusters of Toscana virus meningo-encephalitis in Livorno Province and Elba Island, July-September 2018.

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.

Serum cytokine and chemokine changes during Toscana virus meningitis.

Toscana virus is an important arbovirus causing meningitis and meningoencephalitis in countries around the Mediterranean Sea. While the clinical syndrome and laboratory diagnostic procedures have been well described, less is known about the immune response in Toscana virus meningitis and a possible use of cytokine and chemokine changes for the clinical follow-up of patients. We here characterized serum cytokine and chemokine profiles from 37 patients during the acute and convalescent phase of the infection. Only few serum cytokine/chemokine changes were detected during Toscana virus meningitis. Markedly increased concentrations of IP-10, interferon-α, IL-22, and eotaxin were found in the acute phase. Levels of interferon-α, IL-22, and eotaxin remained elevated in the convalescent phase, but decreased concentrations of GM-CSF were detected.

Meningitis and epididymitis caused by Toscana virus infection imported to Switzerland diagnosed by metagenomic sequencing: a case report.

BACKGROUND: We report a rare case of Toscana virus infection imported into Switzerland in a 23-year old man who travelled to Imperia (Italy) 10 days before onset of symptoms. Symptoms included both meningitis and as well epididymitis. This is only the fourth case of Toscana virus reported in Switzerland. CASE PRESENTATION: The patient presented with lymphocytic meningitis and scrotal pain due to epididymitis. Meningitis was initially treated with ceftriaxone. Herpes simplex, tick-borne encephalitis, enterovirus, measles, mumps, rubella and Treponema pallidum were excluded with specific polymerase chain reaction (PCR) or serology. In support of routine diagnostic PCR and serology assays, unbiased viral metagenomic sequencing was performed of cerebrospinal fluid and serum. Toscana virus infection was identified in cerebrospinal fluid and the full coding sequence could be obtained. Specific PCR in cerebrospinal fluid and blood and serology with Immunoglobulin (Ig) M and IgG against Toscana virus confirmed our diagnosis. Neurological symptoms recovered spontaneously after 5 days. CONCLUSIONS: This case of Toscana virus infection highlights the benefits of unbiased metagenomic sequencing to support routine diagnostics in rare or unexpected viral infections. With increasing travel histories of patients, physicians should be aware of imported Toscana virus as the agent for viral meningitis and meningoencephalitis.

Toscana, West Nile, Usutu and tick-borne encephalitis viruses: external quality assessment for molecular detection of emerging neurotropic viruses in Europe, 2017.

BackgroundNeurotropic arboviruses are increasingly recognised as causative agents of neurological disease in Europe but underdiagnosis is still suspected. Capability for accurate diagnosis is a prerequisite for adequate clinical and public health response.AimTo improve diagnostic capability in EVD-LabNet laboratories, we organised an external quality assessment (EQA) focusing on molecular detection of Toscana (TOSV), Usutu (USUV), West Nile (WNV) and tick-borne encephalitis viruses (TBEV).MethodsSixty-nine laboratories were invited. The EQA panel included two WNV RNA-positive samples (lineages 1 and 2), two TOSV RNA-positive samples (lineages A and B), one TBEV RNA-positive sample (Western subtype), one USUV RNA-positive sample and four negative samples. The EQA focused on overall capability rather than sensitivity of the used techniques. Only detection of one, clinically relevant, concentration per virus species and lineage was assessed.ResultsThe final EQA analysis included 51 laboratories from 35 countries; 44 of these laboratories were from 28 of 31 countries in the European Union/European Economic Area (EU/EEA). USUV diagnostic capability was lowest (28 laboratories in 18 countries), WNV detection capacity was highest (48 laboratories in 32 countries). Twenty-five laboratories were able to test the whole EQA panel, of which only 11 provided completely correct results. The highest scores were observed for WNV and TOSV (92%), followed by TBEV (86%) and USUV (75%).ConclusionWe observed wide variety in extraction methods and RT-PCR tests, showing a profound absence of standardisation across European laboratories. Overall, the results were not satisfactory; capacity and capability need to be improved in 40 laboratories.

High Rates of Neutralizing Antibodies to Toscana and Sandfly Fever Sicilian Viruses in Livestock, Kosovo.

Toscana and sandfly fever Sicilian viruses (TOSV and SFSV, respectively), both transmitted by sand flies, are prominent human pathogens in the Old World. Of 1,086 serum samples collected from cattle and sheep during 2013 in various regions of Kosovo (Balkan Peninsula), 4.7% and 53.4% had neutralizing antibodies against TOSV and SFSV, respectively.

Toscana virus cap-snatching and initiation of transcription.

Toscana virus (TOSV) is an arthropod-borne phlebovirus within the family Phenuiviridae in the order Bunyavirales. It seems to be an important agent of human meningoencephalitis in the warm season in the Mediterranean area. Because the polymerase of Bunyavirales lacks a capping activity, it cleaves short-capped RNA leaders derived from the host cell, and uses them to initiate viral mRNA synthesis. To determine the size and nucleotide composition of the host-derived RNA leaders, and to elucidate the first steps of TOSV transcription initiation, we performed a high-throughput sequencing of the 5' end of TOSV mRNAs in infected cells at different times post-infection. Our results indicated that the viral polymerase cleaved the host-capped RNA leaders within a window of 11-16 nucleotides. A single population of cellular mRNAs could be cleaved at different sites to prime the synthesis of several viral mRNA species. The majority of the mRNA resulted from direct priming, but we observed mRNAs resulting from several rounds of prime-and-realign events. Our data suggest that the different rounds of the prime-and-realign mechanism result from the blocking of the template strand in a static position in the active site, leading to the slippage of the nascent strand by two nucleotides when the growing duplex is sorted out from the active site. To minimize this rate-limiting step, TOSV polymerase cleaves preferentially capped RNA leaders after GC, so as to greatly reduce the number of cycles of priming and realignment, and facilitate the separation of the growing duplex.

Detection of Toscana virus from an adult traveler returning to Australia with encephalitis.

Toscana virus (TOSV) is identified in sandflies, animals, and humans around the Mediterranean Sea. TOSV has not been reported in Australia. During investigations of cerebrospinal fluid samples from patients with encephalitis, TOSV genetic sequences were identified in a traveler returning to Australia from Europe. TOSV should be considered, especially during May to October, in travelers to Australia who embarked in countries in and around the Mediterranean Sea and who subsequently present for medical care because of neurological symptoms.

Toscana virus meningo-encephalitis: an important differential diagnosis for elderly travellers returning from Mediterranean countries.

BACKGROUND: Elderly patients have a long list of differentials for causes of acute confusion and altered consciousness levels, including infectious agents. In addition, elderly, retired patients often have more time to travel for tourism, particularly to exotic, warmer locations. Mediterranean countries such as Spain and Italy are popular holiday destinations for British and other tourists, especially during the winter months. However, these warm climates allow insect vectors to proliferate, increasing the risk of exposure to endemic vectorborne viral infections whilst on vacation. Such infections may not be routinely considered by geriatric medical teams. CASE PRESENTATION: An 87-year old gentleman presented with a three-day history of worsening confusion, lethargy, ataxia, and fevers following a trip to Spain, where he may have sustained a sandfly bite. By the time of admission, he had a reduced GCS, was hallucinating, and was incontinent of urine and faeces, though blood pressure and heart rate were normal. He also appeared hyperaesthetic, and found even capillary blood sugar testing extremely painful. He had no history of cognitive defect or other neurological conditions. He had been previously independently active, with frequent trips to Spain where he maintained a holiday home. He probably sustained a sandfly bite during this most recent trip, whilst cleaning out a shed. Acute and convalescent sera demonstrated IgG antibodies to Toscana virus at extremely high titres of ≥1:10,000 by immunofluorescence assay, though no Toscana virus RNA was detectable in these sera by the time of presentation. CONCLUSIONS: Toscana virus should be included in the differential diagnosis of any patients presenting with meningo-encephalitis who have recently returned from a Mediterranean country. Testing for Toscana virus infection is performed by serological testing on acute/convalescent paired sera, and/or a polymerase chain reaction (PCR) assay on blood or cerebrospinal fluid (CSF) if presenting within 5 days of illness onset. Making a diagnosis of Toscana virus meningitis/encephalitis (where no other pathogen is detected) has additional clinical utility in reducing or preventing unnecessary use of antibiotics, as well as reassuring the patient and family that generally, this illness is generally self-limiting and full recovery within a few weeks is expected, as in the case reported here.

Toscana virus infects dendritic and endothelial cells opening the way for the central nervous system.

Toscana virus (TOSV) is a Phlebovirus responsible for human neurological infections in endemic Mediterranean areas. The main viral target is the central nervous system, with viremia as a way of dissemination throughout the host. This study was aimed at understanding the spread of TOSV in the host by identifying the cell population infected by the virus and the vehicle to the organs. In vivo studies provided evidence that endothelial cells are infected by TOSV, indicating their potential role in the diffusion of the virus following viremic spread. These results were further confirmed in vitro. Human peripheral mononuclear blood cells were infected with TOSV; only monocyte-derived dendritic cells were identified as susceptible to TOSV infection. Productive viral replication was then observed in human monocyte-derived dendritic cells (moDCs) and in human endothelial cells by recovery of the virus from a cell supernatant. Interleukin-6 was produced by both cell types upon TOSV infection, mostly by endothelial cells, while moDCs particularly expressed TNF-α, which is known to induce a long-lasting decrease in endothelial cell barrier function. These cells could therefore be implicated in the spread of the virus in the host and in the infection of tissues that are affected by the disease, such as the central nervous system. The identification of in vitro and in vivo TOSV cell targets is an important tool for understanding the pathogenesis of the infection, providing new insight into virus-cell interaction for improved knowledge and control of this viral disease.