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Toscana virus (TOSV) is classified in the sandfly fever virus group of arboviruses (family Bunyaviridae, genus Phlebovirus) along with Sicilian (SFSV) and Neapolitan (SFNV) sandfly fever virus. SFSV and SFNV cause a transient febrile illness, whereas TOSV also causes mild acute aseptic meningitis. In endemic regions TOSV has been identified as an important cause of viral aseptic meningitis among the indigenous population. TOSV is transmitted by the sandfly species Phlebotomus perniciosus and Phlebotomus perfiliewi. Circulation of TOSV follows the distribution of its vectors—that is, the Mediterranean, Middle East, Western Asia, and North Africa—and the disease coincides with the seasonal life cycle of insect vectors.1,3 Laboratory confirmation of clinically suspected cases can be carried out by detection of anti-TOSV IgG or IgM antibodies in blood and cerebrospinal fluid (CSF) using different techniques. Direct detection of virus is possible by cell culture or reverse transcriptase polymerase chain reaction (RT-PCR) but will often fail because of the short term nature of the viraemia.
Throughout the last decades there have been increasing numbers of cases of TOSV infection imported into northern Europe from endemic areas. Among other causes, it therefore now has to be considered in the differential diagnosis of acute aseptic meningitis.1,2
Four days after having returned from Malaga, Spain, in July 2004 an 80 year old woman was found in a state of deteriorated consciousness. There was enuresis and encopresis, probably as a result of an epileptic fit. Apart from adult onset diabetes mellitus the patient’s medical history was unremarkable.
On admission to hospital, the patient was confused and agitated, with a body temperature of 39.5°C. There was neck rigidity but neurological examination showed no other abnormalities.
Routine clinical chemistry analyses on admission showed slight increases in liver enzymes (aspartate aminotransferase 50 U/l; alanine aminotransferase 87 U/l; γ-glutamyltransferase 93 U/l), erythrocyte sedimentation rate (18 mm/h), and glycated haemoglobin (HbA1c; 7.6%).
Examination of CSF revealed lymphocytic pleocytosis (265 cells/mm3) and raised protein (47 mg/dl) and lactate (4.3 mmol/l) (table 1). PCR for herpes simplex virus 1 (HSV1) and HSV2 was negative.
Both CSF and serum samples collected on days 2 and 6 of disease tested negative for antibodies against West Nile and tick born encephalitis virus by immunofluorescence assay (IFA). RT-PCR for TOSV4 and enterovirus RNA was negative. Infection of Vero cells with CSF yielded no cytopathic effect.
Serum and CSF samples collected on days 2, 6, 13, and 27 after admission were examined for the presence of anti-TOSV, anti-SFNV, and anti-SFSV IgG and IgM antibodies. IgG and IgM IFA5 revealed positive results for both TOSV (strain ISS.Phl.3 1971) and SFNV (strain Sabin 1985), but anti-SFSv (strain Sabin 1985) antibodies were not found in either serum or CSF samples.
Anti-TOSV IgG titres ranged from 1:640 to 1:10240 in serum and 1:20 to 1:5120 in CSF. IgG levels for SFNV ranged from 1:320 to 1:2560 and from 1:20 to 1:1280, respectively. IFA IgM titres could only be detected in serum, ranging from 1:80 to 1:160 for TOSV and from 1:40 to 1:80 for SFNV.
The presence of a pattern of cross reactivity typical of TOSV infection2 supported a preliminary diagnosis of acute TOSV encephalitis. For confirmation, the specificity of antibodies was demonstrated by a commercially available TOSV IgG/IgM enzyme immunosorbent assay (EIA) using recombinant antigen (Diesse Diagnostica, Milan, Italy).5 IFA results for serum samples were confirmed by EIA. Nevertheless, EIA appeared more sensitive than IFA in detecting CSF IgM antibodies (first positive result for CSF IgM in EIA on day 2). In contrast, IgG in CSF samples was only inconsistently detected by EIA (negative results for CSF IgG in EIA on days 2, 13, and 27).
The patient was transferred to the neurological critical care unit and treated empirically with intravenous ceftriaxon, levofloxacin, and aciclovir until diagnosis of TOSV meningoencephalitis was confirmed. Consciousness deteriorated subsequently, leaving the patient in a comatose state for three days. On day three after admission a generalised seizure occurred. Electroencephalography (EEG) showed signs of diffuse encephalopathy with basal activity at 6–7 Hz and symmetrical slow waves. Computed tomography and magnetic resonance imaging of the brain showed diffuse atrophy without evidence of focal lesions. Measurement of TAU protein, S100 protein, and neurone specific enolase in the CSF revealed no signs of neuronal cell damage. Nevertheless, raised β2-microglobulin levels and oligoclonal banding showed that there was an intrathecal immune reaction (table 1).
Ten days after admission the patient recovered from her comatose state. A slow improvement in her level of consciousness and her general condition was observed subsequently as the fever resolved.
One month after admission CSF and liver enzymes were nearly normal. EEG showed an increase of basal activity to 8 c/s. Slightly impaired cognitive functions could still be observed (mini-mental state examination score 17/30).
Besides Rift Valley fever virus, TOSV is the only member of the Phlebovirus group causing aseptic meningitis. Previously, severe neurological manifestations have been described only occasionally. Within three to six days after infection symptoms such as fever, myalgia, headache, vomiting, and neck rigidity do occur, but a lesser degree of encephalic involvement has generally been reported. As in other viral meningitides, symptoms of aseptic meningitis related to TOSV infection disappear completely within a few days. Asymptomatic infections and infections without CNS involvement also occur.1–3
The brain involvement in this case caused a severe, long lasting impairment of consciousness, seizures as an unusual clinical manifestation, and a prolonged convalescence period. Seldom reported before, raised liver enzymes can be interpreted as a sign of a systemic infection with involvement of the liver.
Owing to the large variety of pathogens causing acute aseptic meningitis, clinicians rely on rapid laboratory confirmation of suspected cases by standard methods such as RT-PCR or the plaque reduction neutralisation test (PRNT). Nowadays, PRNT is rarely used owing to its complexity and time consuming nature, while RT-PCR appears inadequate because of the short duration of the viraemia.1,2 Recently developed EIA formats can establish the presence of infection by detecting specific IgM in acute phase samples and were able to confirm TOSV infection in this case. With millions of travellers and increasing TOSV infections around the Mediterranean basin, commercially available EIA methods will be of growing importance. In addition to other severe travel related diseases, TOSV infections now need to be considered by physicians.
Competing interests: none declared