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Extensive Recruitment of Plasma Blasts to the Cerebrospinal Fluid in Toscana Virus Encephalitis

Extensive Recruitment of Plasma Blasts to the Cerebrospinal Fluid in Toscana Virus Encephalitis BRIEF R EP ORT detection of viral RNA is often difficult due to the low concen- Extensive Recruitment of Plasma tration of TOSV-specific nucleic acid in cerebrospinal fluid Blasts to the Cerebrospinal Fluid in (CSF). We describe the case and extensive clinical work-up of a 73- Toscana Virus Encephalitis year-old man with the clinical picture of encephalitis. Informed 1 2 3 1 consent of the patient was obtained for this study. The patient’s Lucas Schirmer, Silke Wölfel, Enrico Georgi, Markus Ploner, 4 1,5 Barbara Bauer, and Bernhard Hemmer symptoms developed gradually over 3 days while he was return- Department of Neurology, Klinikum Rechts der Isar, Technische Universität ing to Germany from vacation on Elba in September 2014. Back 2 3 München, Departments of Virology and Rickettsiology, Microbial Genomics and in Germany, the patient was initially admitted to a local hospital Bioinformatics, Bundeswehr Institute of Microbiology, German Center for Infection with mild fever up to 38°C, headache, myalgia, and episodes of Research (DZIF), Institute of Virology, Technische Universität München and vomiting. As symptoms worsened, he was transferred to our Helmholtz Zentrum München, German Center for Infection Research (DZIF), and Munich Cluster for Systems Neurology (SyNergy), Munich, Germany university medical center, where he presented with signs of confusion, headache, nausea, and photophobia. The patient recovered gradually and had only minor episodes of headaches An unexpectedly extensive recruitment of B cells and plasma blasts to the cerebrospinal fluid (CSF) in a patient with To- 13 days after disease onset. scana virus (TOSV) encephalitis is described. Acute infection Flow cytometry analysis identified a transient and remarkable by TOSV was demonstrated by serological methods and by recruitment of B cells with a large proportion of plasma blasts to detection of TOSV-specific nucleic acid in the CSF by real- the CSF compartment. Acute infection by TOSV was supported time polymerase chain reaction and sequencing. by anti-TOSV serum antibodies with significant titer changes during the course of disease. Central nervous system infection Keywords. B cells; cerebrospinal fluid; encephalitis; plasma blasts; Toscana virus. was demonstrated by TOSV-specific nested polymerase chain reaction (PCR) and sequencing of RNA extracted from the CSF of the patient. In the last years, Toscana virus (TOSV) infection has been iden- MATERIALS AND METHODS tified as an important cause of meningoencephalitis in a sub- group of patients in certain areas of Mediterranean countries Serum and CSF samples were collected on day 5 and 13 after [1, 2]. Toscana virus belongs to the family of Bunyaviridae, disease onset and immediately processed. A follow-up serum genus Phlebovirus, with a trisegmented negative sense RNA ge- sample was obtained 27 days after disease onset. Basic CSF nome [1–3]. In recent studies, TOSV infections in travelers from work-up consisted of microscopic cell counting and May- the Island of Elba, Italy, who developed meningoencephalitis in Grünwald cell stain; measurement of glucose/lactate levels and their home countries have been described [4, 5]. Although the nephelometric protein analysis (albumin, immunoglobulin [Ig] seroprevalence of TOSV antibodies in selected Italian popula- G, IgA, IgM) were done in parallel. tions ranges from 22% to 77%, the reported number of cases For flow cytometry analysis, CSF samples were directly with severe central nervous system (CNS) involvement is limit- washed and processed. Ex vivo staining was performed with an- ed in those areas [6–9]. Serum anti-TOSV antibodies cross-react tibodies against CD45 (clone HI30; BD Biosciences [BD]), CD3 strongly with sandfly fever Naples virus (SFNV), and the (clone SK7; BD), CD19 (clone J3.119; Beckman Coulter [BC]), CD138 (clones B-A38; BC), CD4 (clone SK3; BD), CD8 (clone SFCI21Thy2D3;BC),CD14(cloneMfP9; BC), andCD56 Received 3 July 2015; accepted 17 August 2015. Correspondence: Bernhard Hemmer, MD, Department of Neurology, Klinikum Rechts der Isar, (clone N901; BC). Stained cells were analyzed using an FACS Technische Universität München, Ismaninger Strasse 22, 81675 Munich, Germany (hemmer@ cytometer (CYAN; BC) and FlowJo Software (Tree Star). lrz.tu-muenchen.de). Two-fold dilutions of sera and CSF specimens were subjected Open Forum Infectious Diseases © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases to endpoint titration with starting dilutions of 1:2 (CSF) and Society of America. This is an Open Access article distributed under the terms of the Creative 1:10 (sera), respectively, to determine IgM and IgG titers. Anti- Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/ by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any body detection was done by an indirect immunofluorescence medium, provided the original work is not altered or transformed in any way, and that the work assay (IFA) using a sandfly fever virus and a flavivirus mosaic is properly cited. For commercial re-use, please contact journals.permissions@oup.com. DOI: 10.1093/ofid/ofv124 (Euroimmun). BRIEF REPORT OFID 1 � � Table 1. Protein and Leukocyte Analysis in the CSF at Day 5 and 13 After Disease Onset CSF/Serum Intrathecal IgM Leukocyte + + + + + + + − low + Albumin Ratio Synthesis Count CD3 CD4 CD3 CD8 CD14 CD56 CD19 CD138 CD19 CD138 −3 9 (×10 ) (mg/L) (×10 /L) T Cells T Cells Monocytes NK Cells B Cells Plasma Blasts Day 5 22.4 136.0 (91%) 264 50.5 6.3 2.1 4.9 9 20.2 Day 13 12.5 24.2 (55%) 25 55 27.6 1 6.5 4.4 0.6 Abbreviations: CSF, cerebrospinal fluid; NK, natural killer. + − low + Immune cell subset frequencies given in % of all CSF leukocytes; reference values for CD19 CD138 B cells and CD19 CD138 plasma blasts in non- inflammatory neurological patients: 0.6% (B cells) and 0.1% (plasma blasts) [13]. Viral RNA was extracted from CSF obtained at day 5 using CSF barrier and reduced intrathecal IgM synthesis of 55% + − QIAamp viral RNA Mini Kit (Qiagen) and tested for West (Table 1). This was paralleled by decreased CD19 CD138 Nile virus (WNV) by artus WNV LC real-time reverse- B-cell counts (4.4% of leukocytes) with a remarkable drop of low + transcription (RT)-PCR Kit (Qiagen) according to the manu- CD19 CD138 plasma blasts to 0.6% in the CSF approaching facturer’s instructions. Toscana virus-specificRT-PCRwas normal levels (Figure 1B, Table 1). carried out according to a protocol previously described [10]. In contrast to B cells and plasma blasts, intrathecal CD8 and + + A partial sequence of the S-segment was obtained by nested CD4 T cells increased between day 5 and 13, whereas CD14 PCR. In brief, a 918 base pair (bp) fragment was amplified monocyte frequencies decreased over time; CD56 natural killer from the extracted nucleic acid using the primers TOS 873f cells remained largely stable (Table 1). 5′-ACTgCTCTTTCCACCTTTTgg-3′ and TOS 1791r 5′-ACA Neuroborreliosis, neurosyphilis, and tickborne encephalitis TTgCTCTTgCTTTTCTTgATg-3′. The obtained PCR product as well as infections due to herpes simplex virus, enteroviruses, was subjected to nested PCR with the primers TOS 960f 5′- cytomegalovirus, and Epstein-Barr virus could be ruled out by AgAgTgACAAAgTggCTgCCTAg-3′ and TOS 5′-1741r CAA serological and molecular methods. Bacteriological work-up in- TgCATgggTgAATgAgTTTg-3′. All protocols were carried out cluding testing for meningococcal infection was negative. Thus, using a superscript III high fidelity kit (Life Technologies). antibiotic and antiviral treatment with acyclovir was stopped. The obtained 782 bp fragment was analyzed by gel electropho- Brain magnetic resonance image scanning did not reveal any resis and Sanger sequencing (GATC Biotech). Phylogenetic tree pathological changes or evidence for meningeal enhancement. analysis of a 663 nucleotide (nt) partial small (S) segment se- Based on the recent travel history to Tuscany with reported quence was performed using the maximum likelihood method insect bites, a diagnostic work-up for sandfly infection was car- based on the Tamura-Nei model and 1000 bootstrap replicates ried out. By IFA, high serum antibody titers against TOSV with Mega 5.0 [11,12]. Identical clusters and highly similar con- (IgM: 1:1280; IgG: 1:2560) and SFNV (IgM: 1:1280; IgG: fidence values were obtained by maximum parsimony method 1:2560) were observed on day 5. Serum anti-TOSV antibodies and neighbour-joining method. temporarily increased (IgM: 1:2560; IgG: 1:10240) on day 13 and dropped on day 27 after disease onset (IgM: 1:1280; IgG: 1:5120). In contrast, antibody titers in the CSF against TOSV RESULTS and SFNV dropped significantly from 1:512 (IgM) and 1:256 (IgG) on day 5 to 1:32 (IgM and IgG) on day 13. It is notewor- Cerebrospinal fluid taken 5 days after disease onset revealed thy that negative molecular and serologic test results were ob- moderate pleocytosis (264 white cells/µL) with normal levels tained for acute WNV infection in CSF and sera, respectively. for glucose and lactate (Table 1). Protein analysis revealed Toscana virus-specific RNA could be detected by real-time blood-CSF barrier dysfunction and evidence for a strong intra- RT-PCR from the CSF, and a 782 bp fragment of TOSV was thecal IgM (but not IgG or IgA) synthesis of 91% (Table 1). Ce- successfully amplified by means of nested PCR. Based on the rebrospinal fluid smears showed a dominance of plasmacytoid obtained sequence data, the TOSV could be grouped in geno- lymphocytes (Figure 1A). Flow cytometry analysis demonstrat- + − type A (Tos A) clustering most closely with another Italian ed increased CD19 CD138 B cell numbers (9% of leukocytes; strain (gb|KM275771; Figure 1C) [11]. reference value in noninflammatory neurological patients: low + 0.6%) with an extensive CD19 CD138 plasma blast recruit- ment to the CSF of 20.2% (reference value: 0.1%; Figure 1B, DISCUSSION Table 1)[13]. Thirteen days after disease onset, CSF analysis revealed de- Meningoencephalitis caused by TOSV infection is an important creased cell numbers (25 cells/µL), normalization of blood- differential diagnosis in patients from endemic regions of 2 OFID BRIEF REPORT � � Figure 1. (A) May-Grünwald cell smear stain of cerebrospinal fluid (CSF) obtained 5 days after disease onset shows dominance of plasmacytoid lym- low + phocytes and plasma blasts (scale bar: 50 µm). (B) Flow cytometry analysis of sequential CSF reveals extensive recruitment of CD19 CD138 plasma blasts low + at day 5 after disease onset with dramatic decline during clinical recovery at day 13 (see framed gates for CD19 CD138 cells; frequencies in % of all CSF leukocytes). (C) Phylogenetic tree analysis of a 663 nucleotide (nt) partial S segment sequence by maximum likelihood method based on the Tamura-Nei model and 1000 bootstrap replicates with Mega 5.0; our herein described case Toscana virus DH 140771 Elba 2014 is highlighted in red bold letters (scale bar indicates number of nt substitutions per site). Phlebotomus spp sandflies and needs to be considered in trav- analysis and flow cytometry. We recently reported increased elers presenting with symptoms of viral meningoencephalitis. B cell and plasma blast counts in the CSF of patients with neu- Due to climate changes, there is a risk that the vector will roinfectious diseases compared with multiple sclerosis and move farther North and establish itself in regions of central Eu- other CNS diseases [13, 17]. Accordingly, elevated B cell and rope, which consecutively may lead to the emergence of sandfly plasma blast numbers have been described in the CSF of fever virus infections [14]. It is likely that the majority of TOSV patients with viral meningitis, human immunodeficiency virus infections in travelers returning from endemic regions remain infection, and neuroborreliosis, however not at the high levels undetected because only a limited number of specialized labo- observed in this TOSV patient [13]. Given that a dramatic de- ratories are able to carry out the required diagnostics. Patients cline in CSF plasma blast numbers during the clinical recovery with TOSV meningitis usually show a mild to moderate clinical of the patient was noted, it can be assumed that the detected low + course and resolve without any sequelae. However, a few severe CD19 CD138 cells belong to a group of short-lived plasma cases including encephalitis have been described in the litera- cells that have the ability to strongly proliferate upon initial an- ture [7, 9, 15, 16]. tigen contact with an intense antibody synthesis [17, 18]. In this case, it was possible to demonstrate a moderate pleo- In recent studies of West Nile encephalitis, a strong plasma cytosis with an extensive appearance of B cells and plasma blasts cell pleocytosis in the CSF has been reported by cytological in the CSF during the peak of clinical symptoms by microscopic smear analysis [19]. It is interesting to note that both TOSV BRIEF REPORT OFID 3 � � 5. Sonderegger B, Hachler H, Dobler G, Frei M. Imported aseptic menin- and WNV are arthropod-borne and enveloped RNA viruses gitis due to Toscana virus acquired on the island of Elba, Italy, August that can cause meningoencephalitis in overlapping areas of 2008. Euro Surveill 2009; 14:pii: 19079. Mediterranean countries [14, 20]. 6. Valassina M, Valentini M, Pugliese A, et al. Serological survey of Tosca- na virus infections in a high-risk population in Italy. Clin Diagn Lab Immunol 2003; 10:483–4. CONCLUSIONS 7. Dionisio D, Valassina M, Ciufolini MG, et al. Encephalitis without meningitis due to sandfly fever virus serotype Toscana. Clin Infect In summary, we present a case of meningoencephalitic TOSV Dis 2001; 32:1241–3. 8. Baldelli F, Ciufolini MG, Francisci D, et al. Unusual presentation of life- infection acquired on Elba, Italy. Direct and indirect proof of threatening Toscana virus meningoencephalitis. Clin Infect Dis 2004; the infection was achieved by an extensive diagnostic work- 38:515–20. up. For the first time, it was possible to conduct an immune 9. Bartels S, Boni L, Kretzschmar HA, Heckmann JG. Lethal encephalitis caused by the Toscana virus in an elderly patient. J Neurol 2011; cell subtyping in the CSF in TOSV encephalitis showing an ex- 259:175–7. ceptionally strong intrathecal recruitment of B cells and plasma 10. Pérez-Ruiz M, Collao X, Navarro-Mari JM, Tenorio A. Reverse tran- blasts. Because this has not been observed in other common scription, real-time PCR assay for detection of Toscana virus. J Clin Virol 2007; 39:276–81. viral meningitis patients (eg, due to herpes or enterovirus infec- 11. Baggieri M, Marchi A, Bucci P, et al. Genetic variability of the S segment tion), it seems possible that arthropod-borne RNA viruses such of Toscana virus. Virus Res 2015; 200:35–44. as WNV and TOSV lead to a unique recruitment of B cells to 12. Tamura K, Peterson D, Peterson N, et al. MEGA5: molecular evolu- the CNS. Because these observations are only based on single tionary genetics analysis using maximum likelihood, evolutionary dis- tance, and maximum parsimony methods. Mol Biol Evol 2011; cases, further investigations are necessary to study immune 28:2731–9. cell subsets in WNV and TOSV encephalitis and the mecha- 13. Kowarik MC, Grummel V, Wemlinger S, et al. Immune cell subtyping nism that leads to the specific B-cell recruitment. in the cerebrospinal fluid of patients with neurological diseases. J. Neurol 2014; 261:130–43. 14. Depaquit J, Grandadam M, Fouque F, et al. Arthropod-borne viruses Acknowledgments transmitted by phlebotomine sandflies in Europe: a review. Euro Sur- veill 2010; 15:19507. We thank Verena Grummel for skillful technical assistance. 15. Papa A, Paraforou T, Papakonstantinou I, et al. Severe encephalitis Financial support. This work was supported by the Technische Univer- caused by Toscana virus, Greece. Emerging Infect Dis 2014;20: sität München and the German Ministry of Defense. 1417–9. Potential conflicts of interest. All authors: No reported conflicts. 16. Kuhn J. Toscana virus causing severe meningoencephalitis in an elderly All authors have submitted the ICMJE Form for Disclosure of Potential traveller. J Neurol Neurosurg Psychiatry 2005; 76:1605–6. Conflicts of Interest. 17. Cepok S. Short-lived plasma blasts are the main B cell effector subset during the course of multiple sclerosis. Brain 2005; 128:1667–76. References 18. Auner HW, Beham-Schmid C, Dillon N, Sabbattini P. The life span of 1. Charrel RN, Gallian P, Navarro-Mari JM, et al. Emergence of Toscana short-lived plasma cells is partly determined by a block on activation of virus in Europe. Emerging Infect Dis 2005; 11:1657–63. apoptotic caspases acting in combination with endoplasmic reticulum 2. Charrel RN, Bichaud L, de Lamballerie X. Emergence of Toscana virus stress. Blood 2010; 116:3445–55. in the Mediterranean area. World J Virol 2012; 1:135–41. 19. Carson PJ, Steidler T, Patron R, et al. Plasma cell pleocytosis in cerebro- 3. Valassina M, Cusi MG, Valensin PE. A Mediterranean arbovirus: the spinal fluid in patients with West Nile virus encephalitis. Clin Infect Dis Toscana virus. J Neurovirol 2003; 9:577–83. 2003; 37:e12–5. 4. Gabriel M, Resch C, Günther S, Schmidt-Chanasit J. Toscana virus in- 20. Delbue S, Ferrante P, Mariotto S, et al. Review of West Nile virus epide- fection imported from Elba into Switzerland. Emerging Infect Dis 2010; miology in Italy and report of a case of West Nile virus encephalitis. J 16:1034–6. Neurovirol 2014; 20:437–41. 4 OFID BRIEF REPORT � � http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Open Forum Infectious Diseases Oxford University Press

Extensive Recruitment of Plasma Blasts to the Cerebrospinal Fluid in Toscana Virus Encephalitis

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Abstract

BRIEF R EP ORT detection of viral RNA is often difficult due to the low concen- Extensive Recruitment of Plasma tration of TOSV-specific nucleic acid in cerebrospinal fluid Blasts to the Cerebrospinal Fluid in (CSF). We describe the case and extensive clinical work-up of a 73- Toscana Virus Encephalitis year-old man with the clinical picture of encephalitis. Informed 1 2 3 1 consent of the patient was obtained for this study. The patient’s Lucas Schirmer, Silke Wölfel, Enrico Georgi, Markus Ploner, 4 1,5 Barbara Bauer, and Bernhard Hemmer symptoms developed gradually over 3 days while he was return- Department of Neurology, Klinikum Rechts der Isar, Technische Universität ing to Germany from vacation on Elba in September 2014. Back 2 3 München, Departments of Virology and Rickettsiology, Microbial Genomics and in Germany, the patient was initially admitted to a local hospital Bioinformatics, Bundeswehr Institute of Microbiology, German Center for Infection with mild fever up to 38°C, headache, myalgia, and episodes of Research (DZIF), Institute of Virology, Technische Universität München and vomiting. As symptoms worsened, he was transferred to our Helmholtz Zentrum München, German Center for Infection Research (DZIF), and Munich Cluster for Systems Neurology (SyNergy), Munich, Germany university medical center, where he presented with signs of confusion, headache, nausea, and photophobia. The patient recovered gradually and had only minor episodes of headaches An unexpectedly extensive recruitment of B cells and plasma blasts to the cerebrospinal fluid (CSF) in a patient with To- 13 days after disease onset. scana virus (TOSV) encephalitis is described. Acute infection Flow cytometry analysis identified a transient and remarkable by TOSV was demonstrated by serological methods and by recruitment of B cells with a large proportion of plasma blasts to detection of TOSV-specific nucleic acid in the CSF by real- the CSF compartment. Acute infection by TOSV was supported time polymerase chain reaction and sequencing. by anti-TOSV serum antibodies with significant titer changes during the course of disease. Central nervous system infection Keywords. B cells; cerebrospinal fluid; encephalitis; plasma blasts; Toscana virus. was demonstrated by TOSV-specific nested polymerase chain reaction (PCR) and sequencing of RNA extracted from the CSF of the patient. In the last years, Toscana virus (TOSV) infection has been iden- MATERIALS AND METHODS tified as an important cause of meningoencephalitis in a sub- group of patients in certain areas of Mediterranean countries Serum and CSF samples were collected on day 5 and 13 after [1, 2]. Toscana virus belongs to the family of Bunyaviridae, disease onset and immediately processed. A follow-up serum genus Phlebovirus, with a trisegmented negative sense RNA ge- sample was obtained 27 days after disease onset. Basic CSF nome [1–3]. In recent studies, TOSV infections in travelers from work-up consisted of microscopic cell counting and May- the Island of Elba, Italy, who developed meningoencephalitis in Grünwald cell stain; measurement of glucose/lactate levels and their home countries have been described [4, 5]. Although the nephelometric protein analysis (albumin, immunoglobulin [Ig] seroprevalence of TOSV antibodies in selected Italian popula- G, IgA, IgM) were done in parallel. tions ranges from 22% to 77%, the reported number of cases For flow cytometry analysis, CSF samples were directly with severe central nervous system (CNS) involvement is limit- washed and processed. Ex vivo staining was performed with an- ed in those areas [6–9]. Serum anti-TOSV antibodies cross-react tibodies against CD45 (clone HI30; BD Biosciences [BD]), CD3 strongly with sandfly fever Naples virus (SFNV), and the (clone SK7; BD), CD19 (clone J3.119; Beckman Coulter [BC]), CD138 (clones B-A38; BC), CD4 (clone SK3; BD), CD8 (clone SFCI21Thy2D3;BC),CD14(cloneMfP9; BC), andCD56 Received 3 July 2015; accepted 17 August 2015. Correspondence: Bernhard Hemmer, MD, Department of Neurology, Klinikum Rechts der Isar, (clone N901; BC). Stained cells were analyzed using an FACS Technische Universität München, Ismaninger Strasse 22, 81675 Munich, Germany (hemmer@ cytometer (CYAN; BC) and FlowJo Software (Tree Star). lrz.tu-muenchen.de). Two-fold dilutions of sera and CSF specimens were subjected Open Forum Infectious Diseases © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases to endpoint titration with starting dilutions of 1:2 (CSF) and Society of America. This is an Open Access article distributed under the terms of the Creative 1:10 (sera), respectively, to determine IgM and IgG titers. Anti- Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/ by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any body detection was done by an indirect immunofluorescence medium, provided the original work is not altered or transformed in any way, and that the work assay (IFA) using a sandfly fever virus and a flavivirus mosaic is properly cited. For commercial re-use, please contact journals.permissions@oup.com. DOI: 10.1093/ofid/ofv124 (Euroimmun). BRIEF REPORT OFID 1 � � Table 1. Protein and Leukocyte Analysis in the CSF at Day 5 and 13 After Disease Onset CSF/Serum Intrathecal IgM Leukocyte + + + + + + + − low + Albumin Ratio Synthesis Count CD3 CD4 CD3 CD8 CD14 CD56 CD19 CD138 CD19 CD138 −3 9 (×10 ) (mg/L) (×10 /L) T Cells T Cells Monocytes NK Cells B Cells Plasma Blasts Day 5 22.4 136.0 (91%) 264 50.5 6.3 2.1 4.9 9 20.2 Day 13 12.5 24.2 (55%) 25 55 27.6 1 6.5 4.4 0.6 Abbreviations: CSF, cerebrospinal fluid; NK, natural killer. + − low + Immune cell subset frequencies given in % of all CSF leukocytes; reference values for CD19 CD138 B cells and CD19 CD138 plasma blasts in non- inflammatory neurological patients: 0.6% (B cells) and 0.1% (plasma blasts) [13]. Viral RNA was extracted from CSF obtained at day 5 using CSF barrier and reduced intrathecal IgM synthesis of 55% + − QIAamp viral RNA Mini Kit (Qiagen) and tested for West (Table 1). This was paralleled by decreased CD19 CD138 Nile virus (WNV) by artus WNV LC real-time reverse- B-cell counts (4.4% of leukocytes) with a remarkable drop of low + transcription (RT)-PCR Kit (Qiagen) according to the manu- CD19 CD138 plasma blasts to 0.6% in the CSF approaching facturer’s instructions. Toscana virus-specificRT-PCRwas normal levels (Figure 1B, Table 1). carried out according to a protocol previously described [10]. In contrast to B cells and plasma blasts, intrathecal CD8 and + + A partial sequence of the S-segment was obtained by nested CD4 T cells increased between day 5 and 13, whereas CD14 PCR. In brief, a 918 base pair (bp) fragment was amplified monocyte frequencies decreased over time; CD56 natural killer from the extracted nucleic acid using the primers TOS 873f cells remained largely stable (Table 1). 5′-ACTgCTCTTTCCACCTTTTgg-3′ and TOS 1791r 5′-ACA Neuroborreliosis, neurosyphilis, and tickborne encephalitis TTgCTCTTgCTTTTCTTgATg-3′. The obtained PCR product as well as infections due to herpes simplex virus, enteroviruses, was subjected to nested PCR with the primers TOS 960f 5′- cytomegalovirus, and Epstein-Barr virus could be ruled out by AgAgTgACAAAgTggCTgCCTAg-3′ and TOS 5′-1741r CAA serological and molecular methods. Bacteriological work-up in- TgCATgggTgAATgAgTTTg-3′. All protocols were carried out cluding testing for meningococcal infection was negative. Thus, using a superscript III high fidelity kit (Life Technologies). antibiotic and antiviral treatment with acyclovir was stopped. The obtained 782 bp fragment was analyzed by gel electropho- Brain magnetic resonance image scanning did not reveal any resis and Sanger sequencing (GATC Biotech). Phylogenetic tree pathological changes or evidence for meningeal enhancement. analysis of a 663 nucleotide (nt) partial small (S) segment se- Based on the recent travel history to Tuscany with reported quence was performed using the maximum likelihood method insect bites, a diagnostic work-up for sandfly infection was car- based on the Tamura-Nei model and 1000 bootstrap replicates ried out. By IFA, high serum antibody titers against TOSV with Mega 5.0 [11,12]. Identical clusters and highly similar con- (IgM: 1:1280; IgG: 1:2560) and SFNV (IgM: 1:1280; IgG: fidence values were obtained by maximum parsimony method 1:2560) were observed on day 5. Serum anti-TOSV antibodies and neighbour-joining method. temporarily increased (IgM: 1:2560; IgG: 1:10240) on day 13 and dropped on day 27 after disease onset (IgM: 1:1280; IgG: 1:5120). In contrast, antibody titers in the CSF against TOSV RESULTS and SFNV dropped significantly from 1:512 (IgM) and 1:256 (IgG) on day 5 to 1:32 (IgM and IgG) on day 13. It is notewor- Cerebrospinal fluid taken 5 days after disease onset revealed thy that negative molecular and serologic test results were ob- moderate pleocytosis (264 white cells/µL) with normal levels tained for acute WNV infection in CSF and sera, respectively. for glucose and lactate (Table 1). Protein analysis revealed Toscana virus-specific RNA could be detected by real-time blood-CSF barrier dysfunction and evidence for a strong intra- RT-PCR from the CSF, and a 782 bp fragment of TOSV was thecal IgM (but not IgG or IgA) synthesis of 91% (Table 1). Ce- successfully amplified by means of nested PCR. Based on the rebrospinal fluid smears showed a dominance of plasmacytoid obtained sequence data, the TOSV could be grouped in geno- lymphocytes (Figure 1A). Flow cytometry analysis demonstrat- + − type A (Tos A) clustering most closely with another Italian ed increased CD19 CD138 B cell numbers (9% of leukocytes; strain (gb|KM275771; Figure 1C) [11]. reference value in noninflammatory neurological patients: low + 0.6%) with an extensive CD19 CD138 plasma blast recruit- ment to the CSF of 20.2% (reference value: 0.1%; Figure 1B, DISCUSSION Table 1)[13]. Thirteen days after disease onset, CSF analysis revealed de- Meningoencephalitis caused by TOSV infection is an important creased cell numbers (25 cells/µL), normalization of blood- differential diagnosis in patients from endemic regions of 2 OFID BRIEF REPORT � � Figure 1. (A) May-Grünwald cell smear stain of cerebrospinal fluid (CSF) obtained 5 days after disease onset shows dominance of plasmacytoid lym- low + phocytes and plasma blasts (scale bar: 50 µm). (B) Flow cytometry analysis of sequential CSF reveals extensive recruitment of CD19 CD138 plasma blasts low + at day 5 after disease onset with dramatic decline during clinical recovery at day 13 (see framed gates for CD19 CD138 cells; frequencies in % of all CSF leukocytes). (C) Phylogenetic tree analysis of a 663 nucleotide (nt) partial S segment sequence by maximum likelihood method based on the Tamura-Nei model and 1000 bootstrap replicates with Mega 5.0; our herein described case Toscana virus DH 140771 Elba 2014 is highlighted in red bold letters (scale bar indicates number of nt substitutions per site). Phlebotomus spp sandflies and needs to be considered in trav- analysis and flow cytometry. We recently reported increased elers presenting with symptoms of viral meningoencephalitis. B cell and plasma blast counts in the CSF of patients with neu- Due to climate changes, there is a risk that the vector will roinfectious diseases compared with multiple sclerosis and move farther North and establish itself in regions of central Eu- other CNS diseases [13, 17]. Accordingly, elevated B cell and rope, which consecutively may lead to the emergence of sandfly plasma blast numbers have been described in the CSF of fever virus infections [14]. It is likely that the majority of TOSV patients with viral meningitis, human immunodeficiency virus infections in travelers returning from endemic regions remain infection, and neuroborreliosis, however not at the high levels undetected because only a limited number of specialized labo- observed in this TOSV patient [13]. Given that a dramatic de- ratories are able to carry out the required diagnostics. Patients cline in CSF plasma blast numbers during the clinical recovery with TOSV meningitis usually show a mild to moderate clinical of the patient was noted, it can be assumed that the detected low + course and resolve without any sequelae. However, a few severe CD19 CD138 cells belong to a group of short-lived plasma cases including encephalitis have been described in the litera- cells that have the ability to strongly proliferate upon initial an- ture [7, 9, 15, 16]. tigen contact with an intense antibody synthesis [17, 18]. In this case, it was possible to demonstrate a moderate pleo- In recent studies of West Nile encephalitis, a strong plasma cytosis with an extensive appearance of B cells and plasma blasts cell pleocytosis in the CSF has been reported by cytological in the CSF during the peak of clinical symptoms by microscopic smear analysis [19]. It is interesting to note that both TOSV BRIEF REPORT OFID 3 � � 5. Sonderegger B, Hachler H, Dobler G, Frei M. Imported aseptic menin- and WNV are arthropod-borne and enveloped RNA viruses gitis due to Toscana virus acquired on the island of Elba, Italy, August that can cause meningoencephalitis in overlapping areas of 2008. Euro Surveill 2009; 14:pii: 19079. Mediterranean countries [14, 20]. 6. Valassina M, Valentini M, Pugliese A, et al. Serological survey of Tosca- na virus infections in a high-risk population in Italy. Clin Diagn Lab Immunol 2003; 10:483–4. CONCLUSIONS 7. Dionisio D, Valassina M, Ciufolini MG, et al. Encephalitis without meningitis due to sandfly fever virus serotype Toscana. Clin Infect In summary, we present a case of meningoencephalitic TOSV Dis 2001; 32:1241–3. 8. Baldelli F, Ciufolini MG, Francisci D, et al. Unusual presentation of life- infection acquired on Elba, Italy. Direct and indirect proof of threatening Toscana virus meningoencephalitis. Clin Infect Dis 2004; the infection was achieved by an extensive diagnostic work- 38:515–20. up. For the first time, it was possible to conduct an immune 9. Bartels S, Boni L, Kretzschmar HA, Heckmann JG. Lethal encephalitis caused by the Toscana virus in an elderly patient. J Neurol 2011; cell subtyping in the CSF in TOSV encephalitis showing an ex- 259:175–7. ceptionally strong intrathecal recruitment of B cells and plasma 10. Pérez-Ruiz M, Collao X, Navarro-Mari JM, Tenorio A. Reverse tran- blasts. Because this has not been observed in other common scription, real-time PCR assay for detection of Toscana virus. J Clin Virol 2007; 39:276–81. viral meningitis patients (eg, due to herpes or enterovirus infec- 11. Baggieri M, Marchi A, Bucci P, et al. Genetic variability of the S segment tion), it seems possible that arthropod-borne RNA viruses such of Toscana virus. Virus Res 2015; 200:35–44. as WNV and TOSV lead to a unique recruitment of B cells to 12. Tamura K, Peterson D, Peterson N, et al. MEGA5: molecular evolu- the CNS. Because these observations are only based on single tionary genetics analysis using maximum likelihood, evolutionary dis- tance, and maximum parsimony methods. Mol Biol Evol 2011; cases, further investigations are necessary to study immune 28:2731–9. cell subsets in WNV and TOSV encephalitis and the mecha- 13. Kowarik MC, Grummel V, Wemlinger S, et al. Immune cell subtyping nism that leads to the specific B-cell recruitment. in the cerebrospinal fluid of patients with neurological diseases. J. Neurol 2014; 261:130–43. 14. Depaquit J, Grandadam M, Fouque F, et al. Arthropod-borne viruses Acknowledgments transmitted by phlebotomine sandflies in Europe: a review. Euro Sur- veill 2010; 15:19507. We thank Verena Grummel for skillful technical assistance. 15. Papa A, Paraforou T, Papakonstantinou I, et al. Severe encephalitis Financial support. This work was supported by the Technische Univer- caused by Toscana virus, Greece. Emerging Infect Dis 2014;20: sität München and the German Ministry of Defense. 1417–9. Potential conflicts of interest. All authors: No reported conflicts. 16. Kuhn J. Toscana virus causing severe meningoencephalitis in an elderly All authors have submitted the ICMJE Form for Disclosure of Potential traveller. J Neurol Neurosurg Psychiatry 2005; 76:1605–6. Conflicts of Interest. 17. Cepok S. Short-lived plasma blasts are the main B cell effector subset during the course of multiple sclerosis. Brain 2005; 128:1667–76. References 18. Auner HW, Beham-Schmid C, Dillon N, Sabbattini P. The life span of 1. Charrel RN, Gallian P, Navarro-Mari JM, et al. Emergence of Toscana short-lived plasma cells is partly determined by a block on activation of virus in Europe. Emerging Infect Dis 2005; 11:1657–63. apoptotic caspases acting in combination with endoplasmic reticulum 2. Charrel RN, Bichaud L, de Lamballerie X. Emergence of Toscana virus stress. Blood 2010; 116:3445–55. in the Mediterranean area. World J Virol 2012; 1:135–41. 19. Carson PJ, Steidler T, Patron R, et al. Plasma cell pleocytosis in cerebro- 3. Valassina M, Cusi MG, Valensin PE. A Mediterranean arbovirus: the spinal fluid in patients with West Nile virus encephalitis. Clin Infect Dis Toscana virus. J Neurovirol 2003; 9:577–83. 2003; 37:e12–5. 4. Gabriel M, Resch C, Günther S, Schmidt-Chanasit J. Toscana virus in- 20. Delbue S, Ferrante P, Mariotto S, et al. Review of West Nile virus epide- fection imported from Elba into Switzerland. Emerging Infect Dis 2010; miology in Italy and report of a case of West Nile virus encephalitis. J 16:1034–6. Neurovirol 2014; 20:437–41. 4 OFID BRIEF REPORT � �

Journal

Open Forum Infectious Diseases – Oxford University Press

Published: Sep 1, 2015

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