Urine: A Pitfall for Molecular Detection of Toscana Virus? An Analytical Proof-of-Concept Study.

Toscana virus (TOSV), a sandfly-borne virus, is an important etiological agent in human acute meningitis and meningoencephalitis in the Mediterranean area during the summer. However, the actual number of TOSV infections is underestimated. Laboratory confirmation is necessary because TOSV infection has overlapping clinical features with other neuro-invasive viral infections. Nowadays, the reference test for direct diagnosis in the acute phase of TOSV infection is the PCR based method for detecting TOSV in cerebrospinal fluid and/or plasma, serum, or blood. Although poorly employed, urine is another helpful biological matrix for TOSV detection. Urine is a matrix rich in PCR inhibitors that affect PCR efficiency; consequently, false negatives could be generated. To investigate the potential effect of urine PCR inhibitors on TOSV detection, we compared undiluted and diluted urine using 10-fold series of spiked TOSV. The results showed a significant improvement in TOSV detection performance in diluted urine (1 TCID50 vs. 1 × 104 TCID50 limit of detection and 101.35% vs. 129.62% efficiency, respectively, in diluted and undiluted urine). In conclusion, our data provide preliminary important insights into the use of diluted urine to limit the impact of the inhibitory effects of urine on the detection of TOSV in RT-PCR-based approaches.

Structural flexibility of Toscana virus nucleoprotein in the presence of a single-chain camelid antibody.

Phenuiviridae nucleoprotein is the main structural and functional component of the viral cycle, protecting the viral RNA and mediating the essential replication/transcription processes. The nucleoprotein (N) binds the RNA using its globular core and polymerizes through the N-terminus, which is presented as a highly flexible arm, as demonstrated in this article. The nucleoprotein exists in an `open' or a `closed' conformation. In the case of the closed conformation the flexible N-terminal arm folds over the RNA-binding cleft, preventing RNA adsorption. In the open conformation the arm is extended in such a way that both RNA adsorption and N polymerization are possible. In this article, single-crystal X-ray diffraction and small-angle X-ray scattering were used to study the N protein of Toscana virus complexed with a single-chain camelid antibody (VHH) and it is shown that in the presence of the antibody the nucleoprotein is unable to achieve a functional assembly to form a ribonucleoprotein complex.

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.

The Role of the Leishmania infantum Infected Dogs as a Potential Reservoir Host for Toscana Virus in a Zoonotic Visceral Leishmaniasis Focus of Northern Tunisia.

The role of dogs as reservoir hosts for Toscana virus (TOSV) remains undetermined. This study investigated TOSV and Leishmania infantum infections in one healthy and three infected dogs with Leishmania (A, B, C) following natural exposition to sandfly bites in a focus of zoonotic visceral leishmaniasis (ZVL) located in Northern Tunisia from June to October 2020. At the end of the exposition period, infected and healthy dogs were examined for TOSV and L. infantum infections by xenodiagnosis using a colony of Phlebotomus perniciosus. Pools of freshly engorged P. perniciosus at days 0 and those at days 7 post-feeding were screened for TOSV and L. infantum by nested PCR in the polymerase gene and kinetoplast minicircle DNA, respectively. In the exposure site, P. pernicious is the most abundant sandfly species. The infection rates of sandflies with TOSV and L. infantum were 0.10 and 0.05%, respectively. Leishmania infantum DNA and TOSV RNA were detected in P. perniciosus females fed on dog B and C, respectively. The isolation of TOSV in Vero cells was achieved from two pools containing P. perniciosus fed on dog C. No pathogens were detected in P. perniciosus females fed on dog A and on control dog. We report for the first time the reservoir competence of dog with ZVL in the transmission of TOSV to sandfly vectors in natural settings, in addition to its role as a main reservoir host of L. infantum.

Characterisation of the antiviral RNA interference response to Toscana virus in sand fly cells.

Toscana virus (TOSV) (Bunyavirales, Phenuiviridae, Phlebovirus, Toscana phlebovirus) and other related human pathogenic arboviruses are transmitted by phlebotomine sand flies. TOSV has been reported in nations bordering the Mediterranean Sea among other regions. Infection can result in febrile illness as well as meningitis and encephalitis. Understanding vector-arbovirus interactions is crucial to improving our knowledge of how arboviruses spread, and in this context, immune responses that control viral replication play a significant role. Extensive research has been conducted on mosquito vector immunity against arboviruses, with RNA interference (RNAi) and specifically the exogenous siRNA (exo-siRNA) pathway playing a critical role. However, the antiviral immunity of phlebotomine sand flies is less well understood. Here we were able to show that the exo-siRNA pathway is active in a Phlebotomus papatasi-derived cell line. Following TOSV infection, distinctive 21 nucleotide virus-derived small interfering RNAs (vsiRNAs) were detected. We also identified the exo-siRNA effector Ago2 in this cell line, and silencing its expression rendered the exo-siRNA pathway largely inactive. Thus, our data show that this pathway is active as an antiviral response against a sand fly transmitted bunyavirus, TOSV.

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.

Exposure to Phlebotomus perniciosus sandfly vectors is positively associated with Toscana virus and Leishmania infantum infection in human blood donors in Murcia Region, southeast Spain.

Antibodies against Phlebotomus perniciosus sandfly salivary gland homogenate (SGH) and recombinant protein rSP03B, sandfly-borne Toscana virus (TOSV), Sandfly Fever Sicilian virus (SFSV) and Leishmania, as well as DNA of the latter parasite, were investigated in 670 blood samples from 575 human donors in Murcia Region, southeast Spain, in 2017 and 2018. The estimated SGH and rSP03B seroprevalences were 69% and 88%, respectively, although correlation between test results was relatively low (ρ = 0.39). Similarly, TOSV, SFSV and Leishmania seroprevalences were 26%, 0% and 1%, respectively, and Leishmania PCR prevalence was 2%. Prevalences were significantly greater in 2017, overdispersed and not spatially related to each other although both were positively associated with SGH but not to rSP03B antibody optical densities, questioning the value of the latter as a diagnostic marker for these infections in humans.

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.

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.

Insights into the Evolutionary Origin of Mediterranean Sandfly Fever Viruses.

Sandfly-transmitted phleboviruses (family Phenuiviridae, order Bunyavirales) are associated with febrile illness and infections of the nervous system in humans. These viruses are almost exclusively found in tropical areas of the New World and restricted to semiarid and temperate zones in the Old World. Here, we discovered seven strains of four previously unknown phleboviruses, named Bogoria virus (BOGV), Embossos virus (EMRV), Kiborgoch virus (KBGV), and Perkerra virus (PERV), as well as the recently discovered Ntepes virus, in sandflies collected in the Kenyan Rift Valley. The genomes have a tripartite organization with conserved termini typical of phleboviruses. LOBV, PERV, and EMBV showed low similarity to known phleboviruses, with less than 55% pairwise amino acid identities in the RNA-directed RNA polymerase (RdRp) proteins, and defined a highly diversified monophyletic clade in sister relationship to the sandfly fever Sicilian serocomplex. All three viruses failed to react with sandfly fever Sicilian virus antisera in recombinant immunofluorescence assays (rIFA), suggesting that the viruses belong to a yet-unknown serogroup. In contrast, KBGV was closely related to Toscana virus (84% identity of RdRp proteins) and shared a most recent common ancestor with the clade comprising sandfly fever Naples and Toscana viruses. KBGV reacted with sandfly fever Naples and Toscana virus antisera in rIFA. The genetic diversity of the detected viruses and their phylogenetic positions implies that the Old World sandfly-borne phleboviruses originated from sub-Saharan Africa. Importantly, our findings suggest that diseases associated with sandfly-borne phlebovirus infections may also affect the Kenyan population.IMPORTANCE Studies on the genetic diversity of arthropod-borne viruses circulating in rural regions can provide critical early indications on new emerging viruses essential for global epidemic preparedness. In this study, we describe the discovery of four phleboviruses in sandflies from the Kenyan Rift Valley. The novel viruses are related to the two medically important serocomplexes, sandfly fever Naples and sandfly fever Sicilian, that are associated with febrile illness and neuroinvasive infections and which were previously not known to occur in sub-Saharan Africa. Knowledge on the occurrence of sandfly-borne phleboviruses in Kenya and elsewhere in Africa can help to decipher their contributions in the etiologies of fevers of unknown origin in patients. Our findings on five genetically diverse phleboviruses detected in Kenya suggest that the common ancestor of Old World phleboviruses existed in sub-Saharan Africa, a hot spot for emerging arboviruses.

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.

Novel Toscana Virus Reverse Genetics System Establishes NSs as an Antagonist of Type I Interferon Responses.

The sand fly-borne Toscana virus (TOSV) is the major cause of human meningoencephalitis in the Mediterranean basin during the summer season. In this work, we have developed a T7 RNA polymerase-driven reverse genetics system to recover infectious particles of a lineage B strain of TOSV. The viral protein pattern and growth properties of the rescued virus (rTOSV) were found to be similar to those of the corresponding wild-type (wt) virus. Using this system, we genetically engineered a TOSV mutant lacking expression of the non-structural protein NSs (rTOSVɸNSs). Unlike rTOSV and the wt virus, rTOSVɸNSs was unable to (i) suppress interferon (IFN)-b messenger RNA induction; and (ii) grow efficiently in cells producing IFN-b. Together, our results highlight the importance of NSs for TOSV in evading the IFN response and provide a comprehensive toolbox to investigate the TOSV life cycle in mammalian and insect host cells, including several novel polyclonal antibodies.

Development of a Reverse Genetics System for Toscana Virus (Lineage A).

Toscana virus (TOSV) is a Phlebovirus in the Phenuiviridae family, order Bunyavirales, found in the countries surrounding the Mediterranean. TOSV is an important cause of seasonal acute meningitis and encephalitis within its range. Here, we determined the full sequence of the TOSV strain 1500590, a lineage A virus obtained from an infected patient (Marseille, 2007) and used this in combination with other sequence information to construct functional cDNA plasmids encoding the viral L, M, and S antigenomic sequences under the control of the T7 RNA promoter to recover recombinant viruses. Importantly, resequencing identified two single nucleotide changes to a TOSV reference genome, which, when corrected, restored functionality to the polymerase L and made it possible to recover infectious recombinant TOSV (rTOSV) from cDNA, as well as establish a minigenome system. Using reverse genetics, we produced an NSs-deletant rTOSV and also obtained viruses expressing reporter genes instead of NSs. The availability of such a system assists investigating questions that require genetic manipulation of the viral genome, such as investigations into replication and tropism, and beyond these fundamental aspects, also the development of novel vaccine design strategies.

An update on Toscana virus distribution, genetics, medical and diagnostic aspects.

BACKGROUND: Toscana virus is an arbovirus transmitted by sand flies within the Mediterranean area where it can cause febrile illness and neuroinvasive infections during the seasonal circulation period of the vector. Although it is an important cause of meningitis and encephalitis, it remains a neglected virus with limited published data, as demonstrated by <250 peer-reviewed articles since the 1970s. OBJECTIVE: The last review article on Toscana virus was published in 2012. The aim was to compile peer-reviewed articles to provide an updated review highlighting recent findings to complement previous review articles. SOURCES: PubMed database was searched using the 'Toscana virus' keyword from 2010 to present. A total of 152 articles were retrieved and identified studies were assessed for novel information on virus genetics, and geographic and medical aspects compared with existing knowledge reported in previous review articles. CONTENT: Studies addressing medical, veterinary and entomological aspects have provided evidence that Toscana virus is present in North Africa, in the Balkan Peninsula, and in most of the Mediterranean islands. Besides the two previously recognized genetic lineages, a novel evolutionary lineage has been identified in the Balkan Peninsula. Co-circulation of two genetic lineages has been demonstrated in France, in Turkey and in Croatia. In addition to meningitis and meningo-encephalitis, which have been reported for 40 years, various neuroinvasive forms have been recently reported such as Guillain-Barré syndrome, hydrocephalus, myositis, fasciitis, polymyeloradiculopathy, deafness and facial paralysis. IMPLICATION: Because it is endemic in countries bordering the Mediterranean, physicians should include Toscana virus in the differential diagnosis of patients presenting with febrile illness and/or neurological manifestations.

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.

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 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.

Direct evidence for an expanded circulation area of the recently identified Balkan virus (Sandfly fever Naples virus species) in several countries of the Balkan archipelago.

BACKGROUND: Recently, Balkan virus (BALKV, family Phenuiviridae, genus Phlebovirus) was discovered in sand flies collected in Albania and genetically characterised as a member of the Sandfly fever Naples species complex. To gain knowledge concerning the geographical area where exposure to BALKV exists, entomological surveys were conducted in 2014 and 2015, in Croatia, Bosnia and Herzegovina (BH), Kosovo, Republic of Macedonia and Serbia. RESULTS: A total of 2830 sand flies were trapped during 2014 and 2015 campaigns, and organised as 263 pools. BALKV RNA was detected in four pools from Croatia and in one pool from BH. Phylogenetic relationships were examined using sequences in the S and L RNA segments. Study of the diversity between BALKV sequences from Albania, Croatia and BH showed that Albanian sequences were the most divergent (9-11% [NP]) from the others and that Croatian and BH sequences were grouped (0.9-5.4% [NP]; 0.7-5% [L]). The sand fly infection rate of BALKV was 0.26% in BH and 0.27% in Croatia. Identification of the species content of pools using cox1 and cytb partial regions showed that the five BALKV positive pools contained Phlebotomus neglectus DNA; in four pools, P neglectus was the unique species, whereas P. tobbi DNA was also detected in one pool. CONCLUSIONS: We report here (i) the first direct evidence that the Balkan virus initially described in coastal Albania has a much wider dissemination area than originally believed, (ii) two real-time RT-PCR assays that may be useful for further screening of patients presenting with fever of unknown origin that may be caused by Balkan virus infection, (iii) entomological results suggesting that Balkan virus is likely transmitted by Phlebotomus neglectus, and possibly other sand fly species of the subgenus Larroussius. So far, BALKV has been detected only in sand flies. Whether BALKV can cause disease in humans is unknown and remains to be investigated.

A Snapshot Avian Surveillance Reveals West Nile Virus and Evidence of Wild Birds Participating in Toscana Virus Circulation.

INTRODUCTION: Birds are involved in the epidemiology of several vector-borne viruses, as amplification hosts for viruses, dissemination vehicles for the vectors, and sources of emerging strains in cross-species transmission. Turkey provides diverse habitats for a variety of wild birds and is located along major bird migration routes. This study was undertaken to provide a cross-sectional screening of avian specimens for a spectrum of vector-borne viruses. MATERIALS AND METHODS: The specimens were collected in Hatay province, in the Mediterranean coast of the Anatolian peninsula, located in the convergence zone of the known migration routes. Generic PCR assays were used for the detection of members of Nairovirus, Flavivirus, and Phlebovirus genera of Flaviviridae and Bunyaviridae families. The circulating viruses were characterized via sequencing and selected specimens were inoculated onto Vero cell lines for virus isolation. RESULTS AND DISCUSSION: Specimens from 72 wild birds belonging in 8 orders and 14 species were collected. A total of 158 specimens that comprise 32 sera (20.3%) from 7 species and 126 tissues (79.7%) from 14 species were screened. Eight specimens (8/158, 5%), obtained from 4 individuals (4/72, 5.5%), were positive. West Nile virus (WNV) lineage 1 sequences were characterized in the spleen, heart, and kidney tissues from a lesser spotted eagle (Clanga pomarina), which distinctly clustered from sequences previously identified in Turkey. Toscana virus (TOSV) genotype A and B sequences were identified in brain and kidney tissues from a greater flamingo (Phoenicopterus roseus), a great white pelican (Pelecanus onocrotalus), and a black stork (Ciconia nigra), without successful virus isolation. Partial amino acid sequences of the viral nucleocapsid protein revealed previously unreported substitutions. This study documents the involvement of avians in WNV dispersion in Anatolia as well in TOSV life cycle.

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.