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Multicentre evaluation of central nervous system infections due to Flavi and Phleboviruses in Turkey
Journal of Infection (2012) 65, 343e349
www.elsevierhealth.com/journals/jinf
Multicentre evaluation of central nervous system infections due to Flavi and Phleboviruses in Turkey Koray Ergunay a,*, A. Arzu Sayiner b, Nadine Litzba c, Sabine Lederer d, Remi Charrel e, Petra Kreher c, Durdal Us a, Matthias Niedrig c, Aykut Ozkul f, Gulsen Hascelik a a
Hacettepe University, Faculty of Medicine, Department of Medical Microbiology, 06100 Ankara, Turkey Dokuz Eylul University, Faculty of Medicine, Department of Medical Microbiology, 35340 Izmir, Turkey c Robert Koch Institute, Centre for Biological Safety (ZBS-1), 13353 Berlin, Germany d Euroimmun AG. Tropical Diseases Department, D-23560 Luebeck, Germany e UMR190 "Emergence des Pathologies Virales" (Aix-Marseille University, Institute of Research for Development, EHESP French School of Public Health), 13005 Marseille, France f Ankara University, Faculty of Veterinary Medicine, Department of Virology, 06110 Ankara, Turkey b
Accepted 9 May 2012 Available online 13 June 2012
KEYWORDS Viral encephalitis; Arboviral encephalitis; Central nervous system; West Nile virus; Tick-borne encephalitis virus; Phlebovirus
Summary Objectives: Flavi- and Phleboviruses associated with central nervous system (CNS) infections including West Nile Virus (WNV), Tick-borne Encephalitis Virus (TBEV) and Toscana Virus (TOSV) cause significant morbidity and mortality in humans. In this study, the impact of these agents have been investigated in CNS infections at referral hospitals in two provinces in Turkey, where circulation of these viruses have previously been recognized. Methods: In the study, 258 samples from 126 individuals from Ankara and 113 samples from 108 individuals from Izmir provinces collected in 2010 were included. Viral RNAs were investigated by multiple genus and strain specific primers. Commercial serological assays were employed in screening and reactive results were evaluated with additional assays and by plaque reduction neutralization assay. Results: Two cases of WNV CNS infections, 14 cases of TOSV infections and one TBEV-exposed individual were identified via serological testing. WNV infections in 61 and 56-year old individuals from Ankara presented with fever and encephalitis without skin rash and residual neurologic damage. TOSV-associated cases from both provinces mainly displayed signs of meningitis. TOSV exposure was documented for the first time from Izmir. Conclusions: WNV, TBEV and TOSV infections must be considered in cases of meningoencephalitis of unknown etiology in Turkey. ª 2012 The British Infection Association. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. Hacettepe University Faculty of Medicine, Department of Microbiology and Clinical Microbiology, Morphology Building 3rd floor, 06100 Sihhiye Ankara, Turkey. Tel.: þ90 312 305 15 60; fax: þ90 312 311 52 50. E-mail addresses: [email protected], [email protected] (K. Ergunay). 0163-4453/$36 ª 2012 The British Infection Association. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jinf.2012.05.010
344
Introduction Human infections with vector-borne viruses (also called arboviruses) are among the most important emerging infectious diseases, due to their impact on public health and changing epidemiological features.1 The arboviruses that manifest as central nervous system (CNS) infections are serious and may produce a fatal outcome or permanent neurological sequelae in the affected individuals.2 The Flavivirus genus of the family Flaviviridae consists of a group of highly important human pathogens, many of which possess the capacity to induce a spectrum of CNS diseases in infected hosts, including Japanese encephalitis virus (JEV), Tick-borne encephalitis virus (TBEV), and West Nile virus (WNV).3,4 All flaviviruses circulate in transmission cycles consisting of vertebrate hosts and insect vectors, in which humans mostly act as dead-end hosts.5 Natural cases of human infection usually follow the bite of an infected tick or mosquito, although incidental cases related to other transmission mechanisms, including the use of infected blood products and organ transplants or, in case of TBEV, oral transfer through consumption of unpasteurized milk products have been reported as well.5,6 The family Bunyaviridae consists of more than 150 viruses and 16 serogroups, classified in five genera: Bunyavirus, Hantavirus, Nairovirus, Phlebovirus and Tospovirus. The Phlebovirus genus includes 37 recognized virus species, mostly transmitted to vertebrates by phlebotomine sandflies, which are geographically distributed in Europe, Africa, Central Asia and the Americas.7 In the phlebovirus genus, sandfly fever Naples virus (SFNV), sandfly fever Sicilian virus (SFSV) and antigenically-related strains circulating in Europe have been associated with an acute, influenzalike febrile disease.8 However, Toscana virus (TOSV), a variant of SFNV, have been observed to be among the most frequent viral pathogens involved in aseptic meningitis occurring during summer in France, Italy, Spain and other countries around the Mediterranean, accounting for as high as 81% of the viruses detected.8e10 Although located in an endemic region, data available on Flavi- and Phlebovirus-related CNS infections in Turkey is relatively limited.11 Serologic evidence for WNV and TBEV exposure is well-documented, however, reports of acute infections are rare.11 Recently, cases of TOSV meningoencephalitis and exposure to major SFNV and SFSV serotypes as well as TOSV have been identified.12 The aim of this study was to investigate the presence and impact of WNV, TBEV and TOSVassociated CNS infections in two regions in Turkey.
Materials and methods Setting and samples The study was performed in two university hospitals, Hacettepe University Hospital in Ankara (Central Anatolia, 39 560 N e 32 520 E) and Dokuz Eylul University Hospital in Izmir (Aegean region, Western Anatolia, 38 260 Ne27 090 E) (Fig. 1). Both centres are tertiary care and major referral hospitals of their regions. Ankara is the capital and second most densely-populated city in Turkey (approximate
K. Ergunay et al. population: 4.8 million) and Izmir is the third most densely-populated city (approximate population: 3.9 million) (http://www.turkstat.gov.tr/Start.do). The clinical samples comprise 125 serum and cerebrospinal fluid (CSF) pairs and 8 single CSF samples from Ankara (collected AprileOctober 2010) and 113 CSFs from Izmir (collected JanuaryeDecember 2010) from patients with the preliminary diagnosis of aseptic meningoencephalitis of presumed viral aetiology. Two separate samples were evaluated from 7 and 5 individuals from Ankara and Izmir, respectively. All samples were interpreted as negative for bacterial, mycobacterial and fungal cultures as well as rapid antigen assays. In CSF samples, Polymerase Chain Reaction (PCR) assays for Herpes simplex virus (HSV) type 1/2, Human Herpesvirus 6, Enteroviruses and M. tuberculosis were non-reactive. The samples, obtained within 1e5 days after the onset of symptoms, were stored in aliquots in 20 C and 80 C for future analysis. Clinical history and laboratory data of the patients were retrieved from hospital medical records. The study protocol was approved by local authorities.
WNV, TBEV and phlebovirus molecular testing All samples were subjected to nucleic acid purification and reverse transcription using random hexamers via commercial assays (High Pure Viral Nucleic Acid Kit, Roche Diagnostics, Germany; RevertAid First Strand cDNA Synthesis Kit, Fermentas, Lithuania; Access RT-PCR kit, Promega, Madison, WI, USA) as directed by the manufacturers. All samples were screened for flaviviruses using a real-time reverse transcription (RT) PCR demonstrated to amplify all recognized members of the genus Flavivirus including WNV and TBEV, in addition to in house WNV-specific real-time PCR, targeting the NS5 region, as described.13,14 For the detection of phleboviruses, a variety of primers targeting different genes were employed in independent reactions including a consensus primer set targeting the viral polymerase gene in the L segment of the viral genome and targeting the nucleoprotein gene in S segment of the SFNV complex including TOSV.15,16 All assays were performed in nested or real-time RT-PCR formats where appropriate as previously described.13e16 For nucleic acid testing, cell culture supernatants of Vero cells inoculated with WNV strain NY99-4132 and TOSV isolate ISS.Phl.3 and PS cells inoculated with TBEV strain K23 were processed as described above, as positive controls.
WNV, TBEV and phlebovirus serological testing Screening for TBEV and WNV IgM and IgG class immunoglobulins in all samples were performed via commercial enzymelinked immunosorbent assays (ELISA) and immunofluorescence assays (IFA) (anti-WNV ELISA and IFA, anti-TBEV ELISA and IFA; Euroimmun, Germany). Samples reactive in initial testing were further evaluated via a commercial IFA incorporating antigens of major Flaviviruses including WFV, TBEV, JEV, Yellow Fever Virus (YFV) and Dengue virus serotypes 1e4 (Flavivirus Profile 2 IFA IgM and IgG; Euroimmun, Germany). Specific antibody synthesis in CSF was evaluated for TBEV via a commercial assay (anti-TBEV ELISA IgG in CSF, Euroimmun, Germany) in serum-CSF pairs. Intrathecal synthesis of WNV-specific IgG antibodies were
Flavi and Phlebovirus infections in Turkey
Figure 1
345
Map displaying the provinces in Turkey where the study was performed.
evaluated using 1:2 CSF and 1:404 serum dilutions according to Reiber and Lange as described previously,17 with calculated CSF/serum albumin and globulin indices to compensate for increased diffusion through the bloodebrain barrier.18 CSF/serum quotient of pathogen-specific antibodies over 1.5 were regarded as positive for intrathecal IgG synthesis.17 For pathogenic phleboviruses, all samples were investigated for IgG and IgM via a commercial IFA (Sandfly Fever Virus Mosaic I, Euroimmun, Germany) that enables simultaneous detection of immunoglobulins against four viral serotypes (SFSV, SFNV, TOSV and Sandfly Fever Cyprus Virus, SFCV). All commercial assays were performed and interpreted according to the manufacturers’ instructions. WNV and TBEV reactive samples in the commercial assays were further evaluated via plaque reduction neutralization assay (PRNA) to rule out cross reactions and to confirm specificity of immunoglobulins as described previously.14,19 Briefly, WNV strain NY99-4132 and Vero cells were employed for WNV PRNT. Samples that neutralized 80% of the challenge virus was considered reactive. For TBEV PRNT, PS cells and TBEV strain K23 were used and 90% plaque reducing neutralization titer (NT90) was calculated.19,20 All PRNA experiments were performed in duplicate. Due to the limited amount of available material, PRNA for TOSV or other phleboviruses could not be performed.
Results WNV assays All sera and/or CSF samples (n:371) were negative for WNV using species-specific and pan-flavivirus PCRs. A total of 8 sera (8/125, 6.4%) from Ankara and 5 CSFs from Izmir (5/113, 4.4%)
were reactive in WNV antibody assays. In 8 sera from Ankara, WNV IgG was initially detected employing the commercial ELISA and 6 samples were further confirmed via the commercial IFA (6/8, 75%). WNV IgM was negative in all assays (Table 1). In two of the corresponding CSFs (2/8, 25%), ELISA and IFA reactivity was observed and the calculated CSF/serum quotient of pathogen-specific antibodies indicated WNV antibody synthesis in the CNS (with CSF/serum quotients of 1.54 and 3.77). Six sera and 2 CSF samples were evaluated via WNV PRNT (excluding the 2 ELISA positive and IFA negative sera). Specific antibodies were found in the 2 sera-CSF pairs, also observed as reactive for intrathecal antibody synthesis. In 5 CSF samples from Izmir, 4 displayed WNV IgG antibodies via ELISA and 3 via IFA, whereas in 1 CSF sample, WNV IgM could be detected in both assays. All samples were observed as negative in WNV PRNT (Table 1). Two individuals from Ankara, for whom WNV-specific antibodies were demonstrated in sera and CSF, as well as pathogen specific intrathecal immunoglobulin synthesis in CSF, were diagnosed as confirmed WNV-related CNS disease. The first patient was a 61-year old male from Ankara, who presented with high fever and alteration of consciousness and was admitted to the hospital in late July, 2010. He had no underlying disease other than chronic hypertension and no focal neurological symptom was noted. Initial CSF examination displayed lymphocytic pleocytosis and increased protein levels. The second patient was a 56-year old male resident of Ankara, presented with high fever, headache, malaise and confusion in August 2010. The initial CSF examination demonstrated increased total albumin/ globulin index. Acyclovir and supportive therapy were initiated for both patients. No significant findings in electroencephalogram (EEG) and cranial magnetic
346 Table 1
K. Ergunay et al. Serological assay results for the viruses associated with central nervous system infections investigated in the study. West Nile virus
Location
Samples
Tick-borne encephalitis virus
IgM IgG PRNA IgM reactive reactive confirmed reactive (#/%) (#/%) (positive/ (#/%) tested)
ANKARA (39 56’N - Serum (n:125) 0 32 52’E) CSF (n: 133) 0 CSF (n:113) 1/0.9 IZMIR (38 260 N27 090 E)
8/6.4 2/1.5 5/4.4
2/6 2/2 0/6
4/3.2 0 0
Toscana virusa
IgG reactive (#/%)
PRNA confirmed (positive/ tested)
IgM IgG reactive reactive (#/%) (#/%)
2/1.6 0 3/2.7
1/6 e 0/1
14/11.2 0 2/1.76
10/8 4/3 3/2.7
CSF: cerebrospinal fluid. a Please refer to the text for results of phleboviruses other than Toscana virus.
resonance imaging were observed. Renal functions were within normal limits and no gastrointestinal symptom or any form of skin rash was noted. They were discharged with stable vital signs and no residual neurological or cognitive impairment within two weeks after admission. No epidemiological factor related to WNV exposure could be identified in hospital records.
TBEV assays All sera and/or CSF samples were negative via pan-flavivirus PCR. Six sera (6/125, 4.8%) from Ankara and 3 CSFs from Izmir (3/113, 2.6%) were reactive in serological TBEV assays (Table 1). In 2 samples from Ankara, TBEV IgG positivity was demonstrated via ELISA and IFA whereas in 4 sera, TBEV IgM was detected via ELISA only. TBEV PRNA demonstrated neutralizing antibodies in one IgG positive serum in a 1:60 dilution. However, specific antibody synthesis in the CNS could not be identified in any of the serum-CSF pairs. Three samples from Izmir were IgG reactive in ELISA. All samples were negative for IgM. In 2 of the 3 IgG reactive CSF samples, TBEV PRNA could not be performed due to insufficient material and the remaining sample was observed as negative (Table 1). The individual from Ankara with TBEV neutralizing IgG antibodies in serum was considered as confirmed TBEV exposure. However, due to the lack of specific antibody synthesis in the CNS and negative PCR results, TBEV could not be suggested as the etiological agent responsible for the CNS symptoms observed. This person was a 68-year-old male residing in Ankara without any history of recent history of tick bites, TBEV vaccination or consumption of unpasteurized milk products.
Phlebovirus assays All sera and/or CSF samples were negative for TOSV or other phleboviruses in all PCR protocols employed. From Ankara, a total of 36 sera (36/125, 28.8%) were reactive for phlebovirus/TOSV antibodies. TOSV IgM was demonstrated in 14 (14/125, 11.2%, Table 1) which was accompanied by TOSV IgG in 2 sera (2/14,14.3%). IgG class antibodies against TOSV and/or other phlebovirus serotypes were identified in 21 (21/125, 16.8%) of the reactive sera which is also accompanied by detectable IgGs against the same virus serotypes
in CSF. IgM antibodies against SFSV/SFCV were observed in 3 sera (3/125, 2.4%). In CSFs from Izmir, seroreactivity against TOSV and/or other phlebovirus serotypes were detected in 6 samples (6/113, 5.3%) which include TOSV IgG in 2, TOSV IgM þ IgG in 1 and TOSV þ SFSV IgM in 1 sample (Table 1), as well as SFSV IgG in 2 samples. Twelve individuals from Ankara, in whom TOSV IgM was detected in sera as the only marker of infection and one individual from Izmir with TOSV þ SFSV IgM reactivity in CSF were considered as probable TOSV infections. The clinical and laboratory features of these patients are shown in Table 2.
Discussion Emerging and reemerging vector-borne viral infections of the CNS continue to pose many challenges for the clinicians as well as diagnostic microbiologists and constitute an important public health problem in the affected countries.1,2 In this study, we have aimed to investigate the impact of three major vector-borne viral pathogens affecting CNS (WNV, TBEV and TOSV) in two university referral hospitals in Ankara and Izmir provinces of Turkey, both lying in the endemic zone and evidence for circulation of these agents have previously been revealed.11 In this study, a total of 371 samples comprising 258 samples (125 serum-CSF pairs and 8 CSF, originating from 126 individuals) from Ankara and 113 CSFs (originating from 108 individuals) from Izmir, found negative for common bacterial, fungal and virological causes of CNS infections were evaluated via nucleic acid tests and serological assays. Multiple primer sets targeting different parts of viral genomes were used in nucleic acid testing, in addition to generic, group-specific primers to detect various lineages or virus variants that might be present. All samples were interpreted as negative for viral RNAs. Due to the lack of PCR-positive samples, virus isolation was not attempted. However, serological assays detected reactive samples for all three viruses investigated. For WNV, 6.4% of the sera from Ankara and 5.3% of the CSFs from Izmir demonstrated reactive results for IgM and/or IgG antibodies in initial screening (Table 1). In two individuals from Ankara (2/126, 1.6%), WNV-specific immunoglobulin synthesis in the CNS was also detected during the evaluation of the serum-CSF pairs. Antibody specificity could further be confirmed in
Flavi and Phlebovirus infections in Turkey Table 2
Clinical symptoms and laboratory data of the cases with Toscana virus IgM seroreactivity.
No. Age/gender/place of residence 1 2 3 4 5 6 7 8
9 10 11 12 13
347
48, male, Ankara 28, male, Yozgat
CSF evaluation
Increased protein Pleocytosis, increased protein 56, female, Ankara Normal 21, female, Ankara Pleocytosis 58, female, Ankara Pleocytosis 29, male, Ankara Normal 70, male, Ankara Pleocytosis, increased protein 58, male, Pleocytosis, Kahramanmaras increased protein & glucose 51, male, Ankara Increased protein 28, male, Ankara Normal 33, male, Ankara Normal 29, male, Ankara Increased protein 75, male, Izmir Lymphocytosis
Meningitic Encephalitic Gastrointestinal TOSV RNA TOSV IgM TOSV IgM symptomsa symptomsb symptomsc in serum/CSF in serum in CSF þ þ
e e
e n.i.
e e
þ þ
e e
þ þ e þ e
þ e þ e þ
e e e e þ
e e e e e
þ þ þ þ þ
e e e e e
þ
þ
þ
e
þ
e
þ þ þ þ e
e e e e þ
e e e þ e
e e e e e
þ þ þ þ n.i.
e e e e þ
CSF: cerebrospinal fluid, TOSV: Toscana Virus, n.i.: no information. All patients were negative for TOSV IgG in sera and/or in CSF. a include neck rigidity and/or Kernig signs. b include muscle paresis, alterations of consciousness, tremor and/or cranial nerve involvement. c include nausea/vomiting (Skin rash, lymphadenopathy,hepatic or renal involvement were not observed in any of the cases).
sera and CSF of these individuals via WNV PRNA and the diagnosis of WNV-related CNS disease was supported, while PRNA was non-reactive for the remaining samples. These individuals were residents of Ankara and had no known risk factors for virus exposure. Probable WNV-associated encephalitis in humans was previously suggested in residents of Ankara province in 2009 and could be confirmed in a 62-year old woman.21,22 The symptoms noted in the previous reports are similar with those of the current study, where unspecified febrile episode was followed by symptoms of abrupt-onset mild encephalitis without residual neurological damage. Recently, WNV infection was suggested in an 11-year old girl from Ankara with fever, skin rash and neurologic symptoms, but lacking convincing evidence for serologic confirmation.23 Seroprevalence rates of 0.56e0.86% have been reported in blood donor surveillance efforts from Ankara14,24. WNV infections could not be proven in any of the 108 individuals from Izmir evaluated in this study. WNV exposure have been identified in residents of Aegean region more than two decades ago25 but no data in favor of virus circulation have been available until 2010. In August 2010, the Turkish Ministry of Health announced seven cases of WNV infections affecting residents of various provinces of the Aegean region including Izmir (http://www.saglik. gov.tr/TR/belge/1-10898/eski2yeni.html). Considering that majority of the WNV infections are mild or inapparent and less than 1% of the exposed individuals demonstrate severe disease,26 it can be suggested that human exposure to WNV is occurring in Central Anatolia and Aegean regions of Turkey. TBEV, primarily transmitted by the bite of ticks of the Ixodes family is considered as one of the most important infections of the central nervous system in several European
countries.27 Although recommended for countries with high incidence, TBEV vaccines are not licenced for use in Turkey. The earliest report on TBEV infections from Turkey dates back to 1968, when three patients presenting mainly with meningeal symptoms from Izmir province, had displayed significant increase in TBEV antibody titers between acute and convalescent sera.28 A PRNA-confirmed seroprevalence of 1.21% was announced in 1980 in residents of the region.25 Since 2007, although IgM and/or IgG seroreactivities have been observed in various studies,11 documented TBEV exposure has been a rare event, solely reported from a blood donor residing in Zonguldak province of the Black Sea region.19 Here, we have observed TBEV seroreactivity in 4.8% and 2.7% of the samples from Ankara and Izmir, respectively (Table 1), which further revealed PRNA-confirmed TBEV exposure in a 68-year-old male from Ankara. However, the presence of specific IgG in serum, lacking IgM, viral nucleic acid or immunoglobulin production in CNS strongly suggests that the CNS symptoms of the person is unlikely to be related to TBEV but reflects an exposure from the past. Nevertheless, probable TBEV infections were detected in Ankara in samples collected in 2009,19 which might indicate, along with the current finding, the activity of TBEV or an antigenicallysimilar virus in the region. Only one out of 3 reactive CSF samples from Izmir could be evaluated via PRNA which displayed negative results (Table 1). Interestingly, one of the TBEV IgG reactive individuals had a previous CSF sample included in the study, which had been obtained within the preceding 3e4 weeks and was evaluated as negative for TBEV immunoglobulins. The findings in this individual might represent a seroconversion of TBEV immunoglobulins but are not sufficient to prove a TBEV-related CNS infection. Nevertheless, the current results suggest sporadic TBEV exposure in the study regions.
348 TOSV, a major agent of viral meningoencephalitis observed during summer in Mediterranean countries where the vector Phlebotomus species are present, has also been sought in the study group. TOSV exposure previously was documented in the vicinity of Izmir province29 and from Ankara province.12 Furthermore, acute TOSV infections were also characterized from Ankara region, which provided evidence for the circulating TOSV strains to belong to genotype A,12 prevalent in Italy and some regions in France.16,30 In this study, TOSV seroreactivity was observed in 22 (22/126, 17.5%) and 4 (4/108, 3.7%) individuals from Ankara and Izmir provinces, respectively. A total of 13 individuals with IgM seroreactivity detected in both study centres are considered as probable TOSV infections, since no sample with viral RNA positivity could be revealed and TOSV PRNA could not be performed due to the availability of limited amount of materials from seroreactive individuals. We have previously demonstrated that TOSV IgM persists in a portion of the exposed individuals, thus, IgM detection may not always indicate an acute infection.31 However, considering the symptomatology and laboratory evaluation of the seroreactive cases, TOSV is likely to be the causative agent, despite the lack of PRNA confirmation. In accordance with the previous report from Ankara province,12 TOSV IgM reactive individuals in this study have primarily displayed signs and symptoms of meningitis, including neck rigidity and/or Kernig’s sign. Paresis, alteration of consciousness, tremors or symptoms of cranial nerve involvement were relatively infrequent (Table 2), similar to the clinical presentation in endemic countries8e10 as well as in Ankara province.12 No residual neurological damage was noted for all individuals upon discharge. Interestingly, two individuals with TOSV IgM seroreactivity from Yozgat (Central Anatolia) and Kahramanmaras (Southeast Anatolia) provinces were also identified in the study in addition to the cases from Ankara and Izmir (Table 2). Although evidence for TOSV circulation has not been detected in these provinces, neighbouring regions with virus activity have been revealed.31 Besides TOSV, evidence for exposure to other phlebovirus serotypes such as SFSV and SFNV, primarily associated with self-limited febrile diseases without CNS involvement was also observed in samples from both regions. The major limitation of this study derives from the retrospective design that has been selected to exclude infections associated with more common bacterial, fungal or viral pathogens. In retrospective evaluations, information on clinical follow-up and complete patient history to identify risk factors for vector and/or virus exposure may not always be retained. We were able to identify limited data on the transmission routes or epidemiological factors related to exposure in confirmed/probable infections in this study. Nevertheless, relevant clinical and laboratory data could be retrieved for the majority of the identified cases. Overall, CNS infections with WNV, TBEV and TOSV do not appear to be frequent, where it can be attributed to less then 1% of the cases for WNV and TBEV and w1% for TOSV. As a conclusion, in retrospective evaluation of serum and CSF samples from patients with CNS infections of presumed viral etiology, confirmed WNV and probable TOSV infections, as well as a confirmed TBEV-exposed person were identified in two provinces of Turkey where previous
K. Ergunay et al. reports suggested the circulation of these agents. In the future, these viruses must be considered in differential diagnosis in cases of meningoencephalitis of unknown etiology.
Acknowledgement The ELISA and IFAs employed in this study were kindly provided by Euroimmun SG, Luebeck, Germany. No other assistance or funding was involved in the study.
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349 25. Serter D. Present status of arbovirus sero-epidemiology in the Aegean region of Turkey. In: Vesenjak-Hirjan J, Caliserh C, editors. Arboviruses in the Mediterranean countries. Zbl Bakt Suppl.9. Gustav Fischer Verlag; 1980. p. 155e61. 26. Hayes EB, Gubler DJ. West Nile virus: epidemiology and clinical features of an emerging epidemic in the United States. Annu Rev Med 2006;57:181e94. 27. Charrel RN, Attoui H, Butenko AM, Clegg JC, Deubel V, Frolova T, et al. Tick-borne virus diseases of human interest in Europe. Clin Microbiol Infect 2004;10:1040e55. 28. Serter F. Tick-borne meningo-encephalitis cases in Izmir area. EU Tıp Fak Mec 1968;7:1e13. 29. Ozbel Y, Ertabaklar H, Ciufolini MG, Marchi A, Fiarentino C, Erensoy S et al. Sandfly Fever viruses (Phleboviruses) transmitted by phlebotomus in Turkey. Microbiologica Balkanica. 3rd Balkan Conference of Microbiology Proceedings, 2003: 152e155. 30. Collao X, Palacios G, Sanbonmatsu-Gamez S, Peres-Ruis M, Negredo AI, Navarro-Mari JM, et al. Genetic diversity of Toscana virus. Emerg Infect Dis 2009;15:574e7. 31. Ergunay K, Aydogan S, Ozcebe OI, Cilek EE, Hacioglu S, Karakaya J, et al. Toscana Virus (TOSV) exposure is confirmed in blood donors from Central, North and South/Southeast Anatolia, Turkey. Zoonoses Public Health 2012;59: 148e54.
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Multicentre evaluation of central nervous system infections due to Flavi and Phleboviruses in Turkey Koray Ergunay a,*, A. Arzu Sayiner b, Nadine Litzba c, Sabine Lederer d, Remi Charrel e, Petra Kreher c, Durdal Us a, Matthias Niedrig c, Aykut Ozkul f, Gulsen Hascelik a a
Hacettepe University, Faculty of Medicine, Department of Medical Microbiology, 06100 Ankara, Turkey Dokuz Eylul University, Faculty of Medicine, Department of Medical Microbiology, 35340 Izmir, Turkey c Robert Koch Institute, Centre for Biological Safety (ZBS-1), 13353 Berlin, Germany d Euroimmun AG. Tropical Diseases Department, D-23560 Luebeck, Germany e UMR190 "Emergence des Pathologies Virales" (Aix-Marseille University, Institute of Research for Development, EHESP French School of Public Health), 13005 Marseille, France f Ankara University, Faculty of Veterinary Medicine, Department of Virology, 06110 Ankara, Turkey b
Accepted 9 May 2012 Available online 13 June 2012
KEYWORDS Viral encephalitis; Arboviral encephalitis; Central nervous system; West Nile virus; Tick-borne encephalitis virus; Phlebovirus
Summary Objectives: Flavi- and Phleboviruses associated with central nervous system (CNS) infections including West Nile Virus (WNV), Tick-borne Encephalitis Virus (TBEV) and Toscana Virus (TOSV) cause significant morbidity and mortality in humans. In this study, the impact of these agents have been investigated in CNS infections at referral hospitals in two provinces in Turkey, where circulation of these viruses have previously been recognized. Methods: In the study, 258 samples from 126 individuals from Ankara and 113 samples from 108 individuals from Izmir provinces collected in 2010 were included. Viral RNAs were investigated by multiple genus and strain specific primers. Commercial serological assays were employed in screening and reactive results were evaluated with additional assays and by plaque reduction neutralization assay. Results: Two cases of WNV CNS infections, 14 cases of TOSV infections and one TBEV-exposed individual were identified via serological testing. WNV infections in 61 and 56-year old individuals from Ankara presented with fever and encephalitis without skin rash and residual neurologic damage. TOSV-associated cases from both provinces mainly displayed signs of meningitis. TOSV exposure was documented for the first time from Izmir. Conclusions: WNV, TBEV and TOSV infections must be considered in cases of meningoencephalitis of unknown etiology in Turkey. ª 2012 The British Infection Association. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. Hacettepe University Faculty of Medicine, Department of Microbiology and Clinical Microbiology, Morphology Building 3rd floor, 06100 Sihhiye Ankara, Turkey. Tel.: þ90 312 305 15 60; fax: þ90 312 311 52 50. E-mail addresses: [email protected], [email protected] (K. Ergunay). 0163-4453/$36 ª 2012 The British Infection Association. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jinf.2012.05.010
344
Introduction Human infections with vector-borne viruses (also called arboviruses) are among the most important emerging infectious diseases, due to their impact on public health and changing epidemiological features.1 The arboviruses that manifest as central nervous system (CNS) infections are serious and may produce a fatal outcome or permanent neurological sequelae in the affected individuals.2 The Flavivirus genus of the family Flaviviridae consists of a group of highly important human pathogens, many of which possess the capacity to induce a spectrum of CNS diseases in infected hosts, including Japanese encephalitis virus (JEV), Tick-borne encephalitis virus (TBEV), and West Nile virus (WNV).3,4 All flaviviruses circulate in transmission cycles consisting of vertebrate hosts and insect vectors, in which humans mostly act as dead-end hosts.5 Natural cases of human infection usually follow the bite of an infected tick or mosquito, although incidental cases related to other transmission mechanisms, including the use of infected blood products and organ transplants or, in case of TBEV, oral transfer through consumption of unpasteurized milk products have been reported as well.5,6 The family Bunyaviridae consists of more than 150 viruses and 16 serogroups, classified in five genera: Bunyavirus, Hantavirus, Nairovirus, Phlebovirus and Tospovirus. The Phlebovirus genus includes 37 recognized virus species, mostly transmitted to vertebrates by phlebotomine sandflies, which are geographically distributed in Europe, Africa, Central Asia and the Americas.7 In the phlebovirus genus, sandfly fever Naples virus (SFNV), sandfly fever Sicilian virus (SFSV) and antigenically-related strains circulating in Europe have been associated with an acute, influenzalike febrile disease.8 However, Toscana virus (TOSV), a variant of SFNV, have been observed to be among the most frequent viral pathogens involved in aseptic meningitis occurring during summer in France, Italy, Spain and other countries around the Mediterranean, accounting for as high as 81% of the viruses detected.8e10 Although located in an endemic region, data available on Flavi- and Phlebovirus-related CNS infections in Turkey is relatively limited.11 Serologic evidence for WNV and TBEV exposure is well-documented, however, reports of acute infections are rare.11 Recently, cases of TOSV meningoencephalitis and exposure to major SFNV and SFSV serotypes as well as TOSV have been identified.12 The aim of this study was to investigate the presence and impact of WNV, TBEV and TOSVassociated CNS infections in two regions in Turkey.
Materials and methods Setting and samples The study was performed in two university hospitals, Hacettepe University Hospital in Ankara (Central Anatolia, 39 560 N e 32 520 E) and Dokuz Eylul University Hospital in Izmir (Aegean region, Western Anatolia, 38 260 Ne27 090 E) (Fig. 1). Both centres are tertiary care and major referral hospitals of their regions. Ankara is the capital and second most densely-populated city in Turkey (approximate
K. Ergunay et al. population: 4.8 million) and Izmir is the third most densely-populated city (approximate population: 3.9 million) (http://www.turkstat.gov.tr/Start.do). The clinical samples comprise 125 serum and cerebrospinal fluid (CSF) pairs and 8 single CSF samples from Ankara (collected AprileOctober 2010) and 113 CSFs from Izmir (collected JanuaryeDecember 2010) from patients with the preliminary diagnosis of aseptic meningoencephalitis of presumed viral aetiology. Two separate samples were evaluated from 7 and 5 individuals from Ankara and Izmir, respectively. All samples were interpreted as negative for bacterial, mycobacterial and fungal cultures as well as rapid antigen assays. In CSF samples, Polymerase Chain Reaction (PCR) assays for Herpes simplex virus (HSV) type 1/2, Human Herpesvirus 6, Enteroviruses and M. tuberculosis were non-reactive. The samples, obtained within 1e5 days after the onset of symptoms, were stored in aliquots in 20 C and 80 C for future analysis. Clinical history and laboratory data of the patients were retrieved from hospital medical records. The study protocol was approved by local authorities.
WNV, TBEV and phlebovirus molecular testing All samples were subjected to nucleic acid purification and reverse transcription using random hexamers via commercial assays (High Pure Viral Nucleic Acid Kit, Roche Diagnostics, Germany; RevertAid First Strand cDNA Synthesis Kit, Fermentas, Lithuania; Access RT-PCR kit, Promega, Madison, WI, USA) as directed by the manufacturers. All samples were screened for flaviviruses using a real-time reverse transcription (RT) PCR demonstrated to amplify all recognized members of the genus Flavivirus including WNV and TBEV, in addition to in house WNV-specific real-time PCR, targeting the NS5 region, as described.13,14 For the detection of phleboviruses, a variety of primers targeting different genes were employed in independent reactions including a consensus primer set targeting the viral polymerase gene in the L segment of the viral genome and targeting the nucleoprotein gene in S segment of the SFNV complex including TOSV.15,16 All assays were performed in nested or real-time RT-PCR formats where appropriate as previously described.13e16 For nucleic acid testing, cell culture supernatants of Vero cells inoculated with WNV strain NY99-4132 and TOSV isolate ISS.Phl.3 and PS cells inoculated with TBEV strain K23 were processed as described above, as positive controls.
WNV, TBEV and phlebovirus serological testing Screening for TBEV and WNV IgM and IgG class immunoglobulins in all samples were performed via commercial enzymelinked immunosorbent assays (ELISA) and immunofluorescence assays (IFA) (anti-WNV ELISA and IFA, anti-TBEV ELISA and IFA; Euroimmun, Germany). Samples reactive in initial testing were further evaluated via a commercial IFA incorporating antigens of major Flaviviruses including WFV, TBEV, JEV, Yellow Fever Virus (YFV) and Dengue virus serotypes 1e4 (Flavivirus Profile 2 IFA IgM and IgG; Euroimmun, Germany). Specific antibody synthesis in CSF was evaluated for TBEV via a commercial assay (anti-TBEV ELISA IgG in CSF, Euroimmun, Germany) in serum-CSF pairs. Intrathecal synthesis of WNV-specific IgG antibodies were
Flavi and Phlebovirus infections in Turkey
Figure 1
345
Map displaying the provinces in Turkey where the study was performed.
evaluated using 1:2 CSF and 1:404 serum dilutions according to Reiber and Lange as described previously,17 with calculated CSF/serum albumin and globulin indices to compensate for increased diffusion through the bloodebrain barrier.18 CSF/serum quotient of pathogen-specific antibodies over 1.5 were regarded as positive for intrathecal IgG synthesis.17 For pathogenic phleboviruses, all samples were investigated for IgG and IgM via a commercial IFA (Sandfly Fever Virus Mosaic I, Euroimmun, Germany) that enables simultaneous detection of immunoglobulins against four viral serotypes (SFSV, SFNV, TOSV and Sandfly Fever Cyprus Virus, SFCV). All commercial assays were performed and interpreted according to the manufacturers’ instructions. WNV and TBEV reactive samples in the commercial assays were further evaluated via plaque reduction neutralization assay (PRNA) to rule out cross reactions and to confirm specificity of immunoglobulins as described previously.14,19 Briefly, WNV strain NY99-4132 and Vero cells were employed for WNV PRNT. Samples that neutralized 80% of the challenge virus was considered reactive. For TBEV PRNT, PS cells and TBEV strain K23 were used and 90% plaque reducing neutralization titer (NT90) was calculated.19,20 All PRNA experiments were performed in duplicate. Due to the limited amount of available material, PRNA for TOSV or other phleboviruses could not be performed.
Results WNV assays All sera and/or CSF samples (n:371) were negative for WNV using species-specific and pan-flavivirus PCRs. A total of 8 sera (8/125, 6.4%) from Ankara and 5 CSFs from Izmir (5/113, 4.4%)
were reactive in WNV antibody assays. In 8 sera from Ankara, WNV IgG was initially detected employing the commercial ELISA and 6 samples were further confirmed via the commercial IFA (6/8, 75%). WNV IgM was negative in all assays (Table 1). In two of the corresponding CSFs (2/8, 25%), ELISA and IFA reactivity was observed and the calculated CSF/serum quotient of pathogen-specific antibodies indicated WNV antibody synthesis in the CNS (with CSF/serum quotients of 1.54 and 3.77). Six sera and 2 CSF samples were evaluated via WNV PRNT (excluding the 2 ELISA positive and IFA negative sera). Specific antibodies were found in the 2 sera-CSF pairs, also observed as reactive for intrathecal antibody synthesis. In 5 CSF samples from Izmir, 4 displayed WNV IgG antibodies via ELISA and 3 via IFA, whereas in 1 CSF sample, WNV IgM could be detected in both assays. All samples were observed as negative in WNV PRNT (Table 1). Two individuals from Ankara, for whom WNV-specific antibodies were demonstrated in sera and CSF, as well as pathogen specific intrathecal immunoglobulin synthesis in CSF, were diagnosed as confirmed WNV-related CNS disease. The first patient was a 61-year old male from Ankara, who presented with high fever and alteration of consciousness and was admitted to the hospital in late July, 2010. He had no underlying disease other than chronic hypertension and no focal neurological symptom was noted. Initial CSF examination displayed lymphocytic pleocytosis and increased protein levels. The second patient was a 56-year old male resident of Ankara, presented with high fever, headache, malaise and confusion in August 2010. The initial CSF examination demonstrated increased total albumin/ globulin index. Acyclovir and supportive therapy were initiated for both patients. No significant findings in electroencephalogram (EEG) and cranial magnetic
346 Table 1
K. Ergunay et al. Serological assay results for the viruses associated with central nervous system infections investigated in the study. West Nile virus
Location
Samples
Tick-borne encephalitis virus
IgM IgG PRNA IgM reactive reactive confirmed reactive (#/%) (#/%) (positive/ (#/%) tested)
ANKARA (39 56’N - Serum (n:125) 0 32 52’E) CSF (n: 133) 0 CSF (n:113) 1/0.9 IZMIR (38 260 N27 090 E)
8/6.4 2/1.5 5/4.4
2/6 2/2 0/6
4/3.2 0 0
Toscana virusa
IgG reactive (#/%)
PRNA confirmed (positive/ tested)
IgM IgG reactive reactive (#/%) (#/%)
2/1.6 0 3/2.7
1/6 e 0/1
14/11.2 0 2/1.76
10/8 4/3 3/2.7
CSF: cerebrospinal fluid. a Please refer to the text for results of phleboviruses other than Toscana virus.
resonance imaging were observed. Renal functions were within normal limits and no gastrointestinal symptom or any form of skin rash was noted. They were discharged with stable vital signs and no residual neurological or cognitive impairment within two weeks after admission. No epidemiological factor related to WNV exposure could be identified in hospital records.
TBEV assays All sera and/or CSF samples were negative via pan-flavivirus PCR. Six sera (6/125, 4.8%) from Ankara and 3 CSFs from Izmir (3/113, 2.6%) were reactive in serological TBEV assays (Table 1). In 2 samples from Ankara, TBEV IgG positivity was demonstrated via ELISA and IFA whereas in 4 sera, TBEV IgM was detected via ELISA only. TBEV PRNA demonstrated neutralizing antibodies in one IgG positive serum in a 1:60 dilution. However, specific antibody synthesis in the CNS could not be identified in any of the serum-CSF pairs. Three samples from Izmir were IgG reactive in ELISA. All samples were negative for IgM. In 2 of the 3 IgG reactive CSF samples, TBEV PRNA could not be performed due to insufficient material and the remaining sample was observed as negative (Table 1). The individual from Ankara with TBEV neutralizing IgG antibodies in serum was considered as confirmed TBEV exposure. However, due to the lack of specific antibody synthesis in the CNS and negative PCR results, TBEV could not be suggested as the etiological agent responsible for the CNS symptoms observed. This person was a 68-year-old male residing in Ankara without any history of recent history of tick bites, TBEV vaccination or consumption of unpasteurized milk products.
Phlebovirus assays All sera and/or CSF samples were negative for TOSV or other phleboviruses in all PCR protocols employed. From Ankara, a total of 36 sera (36/125, 28.8%) were reactive for phlebovirus/TOSV antibodies. TOSV IgM was demonstrated in 14 (14/125, 11.2%, Table 1) which was accompanied by TOSV IgG in 2 sera (2/14,14.3%). IgG class antibodies against TOSV and/or other phlebovirus serotypes were identified in 21 (21/125, 16.8%) of the reactive sera which is also accompanied by detectable IgGs against the same virus serotypes
in CSF. IgM antibodies against SFSV/SFCV were observed in 3 sera (3/125, 2.4%). In CSFs from Izmir, seroreactivity against TOSV and/or other phlebovirus serotypes were detected in 6 samples (6/113, 5.3%) which include TOSV IgG in 2, TOSV IgM þ IgG in 1 and TOSV þ SFSV IgM in 1 sample (Table 1), as well as SFSV IgG in 2 samples. Twelve individuals from Ankara, in whom TOSV IgM was detected in sera as the only marker of infection and one individual from Izmir with TOSV þ SFSV IgM reactivity in CSF were considered as probable TOSV infections. The clinical and laboratory features of these patients are shown in Table 2.
Discussion Emerging and reemerging vector-borne viral infections of the CNS continue to pose many challenges for the clinicians as well as diagnostic microbiologists and constitute an important public health problem in the affected countries.1,2 In this study, we have aimed to investigate the impact of three major vector-borne viral pathogens affecting CNS (WNV, TBEV and TOSV) in two university referral hospitals in Ankara and Izmir provinces of Turkey, both lying in the endemic zone and evidence for circulation of these agents have previously been revealed.11 In this study, a total of 371 samples comprising 258 samples (125 serum-CSF pairs and 8 CSF, originating from 126 individuals) from Ankara and 113 CSFs (originating from 108 individuals) from Izmir, found negative for common bacterial, fungal and virological causes of CNS infections were evaluated via nucleic acid tests and serological assays. Multiple primer sets targeting different parts of viral genomes were used in nucleic acid testing, in addition to generic, group-specific primers to detect various lineages or virus variants that might be present. All samples were interpreted as negative for viral RNAs. Due to the lack of PCR-positive samples, virus isolation was not attempted. However, serological assays detected reactive samples for all three viruses investigated. For WNV, 6.4% of the sera from Ankara and 5.3% of the CSFs from Izmir demonstrated reactive results for IgM and/or IgG antibodies in initial screening (Table 1). In two individuals from Ankara (2/126, 1.6%), WNV-specific immunoglobulin synthesis in the CNS was also detected during the evaluation of the serum-CSF pairs. Antibody specificity could further be confirmed in
Flavi and Phlebovirus infections in Turkey Table 2
Clinical symptoms and laboratory data of the cases with Toscana virus IgM seroreactivity.
No. Age/gender/place of residence 1 2 3 4 5 6 7 8
9 10 11 12 13
347
48, male, Ankara 28, male, Yozgat
CSF evaluation
Increased protein Pleocytosis, increased protein 56, female, Ankara Normal 21, female, Ankara Pleocytosis 58, female, Ankara Pleocytosis 29, male, Ankara Normal 70, male, Ankara Pleocytosis, increased protein 58, male, Pleocytosis, Kahramanmaras increased protein & glucose 51, male, Ankara Increased protein 28, male, Ankara Normal 33, male, Ankara Normal 29, male, Ankara Increased protein 75, male, Izmir Lymphocytosis
Meningitic Encephalitic Gastrointestinal TOSV RNA TOSV IgM TOSV IgM symptomsa symptomsb symptomsc in serum/CSF in serum in CSF þ þ
e e
e n.i.
e e
þ þ
e e
þ þ e þ e
þ e þ e þ
e e e e þ
e e e e e
þ þ þ þ þ
e e e e e
þ
þ
þ
e
þ
e
þ þ þ þ e
e e e e þ
e e e þ e
e e e e e
þ þ þ þ n.i.
e e e e þ
CSF: cerebrospinal fluid, TOSV: Toscana Virus, n.i.: no information. All patients were negative for TOSV IgG in sera and/or in CSF. a include neck rigidity and/or Kernig signs. b include muscle paresis, alterations of consciousness, tremor and/or cranial nerve involvement. c include nausea/vomiting (Skin rash, lymphadenopathy,hepatic or renal involvement were not observed in any of the cases).
sera and CSF of these individuals via WNV PRNA and the diagnosis of WNV-related CNS disease was supported, while PRNA was non-reactive for the remaining samples. These individuals were residents of Ankara and had no known risk factors for virus exposure. Probable WNV-associated encephalitis in humans was previously suggested in residents of Ankara province in 2009 and could be confirmed in a 62-year old woman.21,22 The symptoms noted in the previous reports are similar with those of the current study, where unspecified febrile episode was followed by symptoms of abrupt-onset mild encephalitis without residual neurological damage. Recently, WNV infection was suggested in an 11-year old girl from Ankara with fever, skin rash and neurologic symptoms, but lacking convincing evidence for serologic confirmation.23 Seroprevalence rates of 0.56e0.86% have been reported in blood donor surveillance efforts from Ankara14,24. WNV infections could not be proven in any of the 108 individuals from Izmir evaluated in this study. WNV exposure have been identified in residents of Aegean region more than two decades ago25 but no data in favor of virus circulation have been available until 2010. In August 2010, the Turkish Ministry of Health announced seven cases of WNV infections affecting residents of various provinces of the Aegean region including Izmir (http://www.saglik. gov.tr/TR/belge/1-10898/eski2yeni.html). Considering that majority of the WNV infections are mild or inapparent and less than 1% of the exposed individuals demonstrate severe disease,26 it can be suggested that human exposure to WNV is occurring in Central Anatolia and Aegean regions of Turkey. TBEV, primarily transmitted by the bite of ticks of the Ixodes family is considered as one of the most important infections of the central nervous system in several European
countries.27 Although recommended for countries with high incidence, TBEV vaccines are not licenced for use in Turkey. The earliest report on TBEV infections from Turkey dates back to 1968, when three patients presenting mainly with meningeal symptoms from Izmir province, had displayed significant increase in TBEV antibody titers between acute and convalescent sera.28 A PRNA-confirmed seroprevalence of 1.21% was announced in 1980 in residents of the region.25 Since 2007, although IgM and/or IgG seroreactivities have been observed in various studies,11 documented TBEV exposure has been a rare event, solely reported from a blood donor residing in Zonguldak province of the Black Sea region.19 Here, we have observed TBEV seroreactivity in 4.8% and 2.7% of the samples from Ankara and Izmir, respectively (Table 1), which further revealed PRNA-confirmed TBEV exposure in a 68-year-old male from Ankara. However, the presence of specific IgG in serum, lacking IgM, viral nucleic acid or immunoglobulin production in CNS strongly suggests that the CNS symptoms of the person is unlikely to be related to TBEV but reflects an exposure from the past. Nevertheless, probable TBEV infections were detected in Ankara in samples collected in 2009,19 which might indicate, along with the current finding, the activity of TBEV or an antigenicallysimilar virus in the region. Only one out of 3 reactive CSF samples from Izmir could be evaluated via PRNA which displayed negative results (Table 1). Interestingly, one of the TBEV IgG reactive individuals had a previous CSF sample included in the study, which had been obtained within the preceding 3e4 weeks and was evaluated as negative for TBEV immunoglobulins. The findings in this individual might represent a seroconversion of TBEV immunoglobulins but are not sufficient to prove a TBEV-related CNS infection. Nevertheless, the current results suggest sporadic TBEV exposure in the study regions.
348 TOSV, a major agent of viral meningoencephalitis observed during summer in Mediterranean countries where the vector Phlebotomus species are present, has also been sought in the study group. TOSV exposure previously was documented in the vicinity of Izmir province29 and from Ankara province.12 Furthermore, acute TOSV infections were also characterized from Ankara region, which provided evidence for the circulating TOSV strains to belong to genotype A,12 prevalent in Italy and some regions in France.16,30 In this study, TOSV seroreactivity was observed in 22 (22/126, 17.5%) and 4 (4/108, 3.7%) individuals from Ankara and Izmir provinces, respectively. A total of 13 individuals with IgM seroreactivity detected in both study centres are considered as probable TOSV infections, since no sample with viral RNA positivity could be revealed and TOSV PRNA could not be performed due to the availability of limited amount of materials from seroreactive individuals. We have previously demonstrated that TOSV IgM persists in a portion of the exposed individuals, thus, IgM detection may not always indicate an acute infection.31 However, considering the symptomatology and laboratory evaluation of the seroreactive cases, TOSV is likely to be the causative agent, despite the lack of PRNA confirmation. In accordance with the previous report from Ankara province,12 TOSV IgM reactive individuals in this study have primarily displayed signs and symptoms of meningitis, including neck rigidity and/or Kernig’s sign. Paresis, alteration of consciousness, tremors or symptoms of cranial nerve involvement were relatively infrequent (Table 2), similar to the clinical presentation in endemic countries8e10 as well as in Ankara province.12 No residual neurological damage was noted for all individuals upon discharge. Interestingly, two individuals with TOSV IgM seroreactivity from Yozgat (Central Anatolia) and Kahramanmaras (Southeast Anatolia) provinces were also identified in the study in addition to the cases from Ankara and Izmir (Table 2). Although evidence for TOSV circulation has not been detected in these provinces, neighbouring regions with virus activity have been revealed.31 Besides TOSV, evidence for exposure to other phlebovirus serotypes such as SFSV and SFNV, primarily associated with self-limited febrile diseases without CNS involvement was also observed in samples from both regions. The major limitation of this study derives from the retrospective design that has been selected to exclude infections associated with more common bacterial, fungal or viral pathogens. In retrospective evaluations, information on clinical follow-up and complete patient history to identify risk factors for vector and/or virus exposure may not always be retained. We were able to identify limited data on the transmission routes or epidemiological factors related to exposure in confirmed/probable infections in this study. Nevertheless, relevant clinical and laboratory data could be retrieved for the majority of the identified cases. Overall, CNS infections with WNV, TBEV and TOSV do not appear to be frequent, where it can be attributed to less then 1% of the cases for WNV and TBEV and w1% for TOSV. As a conclusion, in retrospective evaluation of serum and CSF samples from patients with CNS infections of presumed viral etiology, confirmed WNV and probable TOSV infections, as well as a confirmed TBEV-exposed person were identified in two provinces of Turkey where previous
K. Ergunay et al. reports suggested the circulation of these agents. In the future, these viruses must be considered in differential diagnosis in cases of meningoencephalitis of unknown etiology.
Acknowledgement The ELISA and IFAs employed in this study were kindly provided by Euroimmun SG, Luebeck, Germany. No other assistance or funding was involved in the study.
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