Report of the Meningitis Program of the International Scientific Working Group on TBE

Vaccine 21 (2003) S1/66–S1/72

Report of the Meningitis Program of the International Scientific Working Group on TBE Serological screening of patients with viral CNS-infection of unknown etiology in search of undiagnosed TBE cases Mats Haglund a,b,∗ , Bo Settergren b,c , Franz X. Heinz d , Göran Günther e , The ISW–TBE Study Group 1 a Department of Infectious Diseases, Kalmar County Hospital, SE-391 85 Kalmar, Sweden Research Institute of Zoonotic Ecology and Epidemiology (RIZEE), Ölands Skogsby 6280, SE-386 93 Färjestaden, Sweden c Department of Infectious Diseases, Central Hospital, S-291 85 Kristianstad, Sweden d Institute of Virology, University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria Clinic of Infectious Diseases, Central Hospital and Center of Clinical Research, Uppsala University, SE-721 89 Västerås, Sweden b

e

Abstract The endemicity of tick-borne encephalitis (TBE) in Europe is changing. Potential undetected or emerging TBEV foci and the risk of underdiagnosis due to a low awareness among the medical community form the background of this retrospective multicenter follow-up study. We investigated the possibility of undiagnosed TBE cases among patients with presumed viral central nervous system (CNS)-infection of unknown etiology. Eight centers in four European countries provided sera and/or cerebrospinal fluid (CSF) samples from 233 individuals. The samples were screened with a commercial TBEV ELISA test system (IgM and IgG). Positive or borderline samples were re-evaluated at the Institute of Virology in Vienna by an in-house ELISA test and a neutralization test (NT). Two previously undiagnosed Swedish TBE patients were verified. Three additional individuals from Swedish centers were IgG ELISA and NT positive. No NT positive individuals were found from France, Belgium or The Netherlands. Nineteen individuals were found IgG TBE ELISA positive, but negative in NT, indicating unspecific reactivity. At least four of those patients were vaccinated against yellow fever. The probable reason for the reactivity seen in these individuals is the well-known cross-reactivity existing among flaviviruses. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Viral CNS-infection; Etiology; Undiagnosed TBE cases; Human

1. Introduction Tick-borne encephalitis (TBE) is an important arthropodborne viral infection in Europe with approximately 3000 confirmed human cases annually [1,2]. Tick-borne encephalitis virus (TBEV) causes infection of the central nervous system (CNS) with a spectrum of clinical presentations, ranging from mild meningitis to severe encephalitis with or without myelitis. A large proportion of the patients is left with permanent sequel [3–6]. In the various coun∗

Corresponding author. E-mail address: [email protected] (M. Haglund). 1 Principal investigator: Mats Haglund; Study committee: Mats Haglund, Göran Günther, Franz X Heinz, Bo Settergren; Investigators: Sweden: Göran Günther, Åke Lundkvist, Bo Settergren; Marie Studahl, Sirkka Vene; France: Alain Coaquette, Fabienne Coquet, Tahar Hadou, Bruno Hoen, Alain Le Faou, Patrick Pl´esiat, H´el´ene Schuhmacher; Belgium: Patrick De Mol; The Netherlands: Wim van der Poel.

tries where TBE is endemic, the possibility for diagnosis is influenced by several factors, such as the availability of serological test systems, the use and quality of the diagnostic tools, regulations, socioeconomic factors, clinical practice based on tradition and previous knowledge of TBE endemicity. Epidemiological surveillance in the endemic countries is based on different reporting systems, providing good coverage of the confirmed cases. Even though coverage has probably not been complete, the information collected over the years allows for a good description of the endemic situation of TBE in Europe, including the Baltic States [1,2]. During the past 10 years, there have been reports describing a decreased incidence in some regions (Thuringia in central Germany, some southern countries as Croatia, Slovenia and Hungary) and an extended endemicity in others (Switzerland, southern Germany) [1,7–10]. These trends are difficult to analyze because the dynamics of TBEV are probably subject to multivariate constraints,

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M. Haglund et al. / Vaccine 21 (2003) S1/66–S1/72

such as climatic, vegetational and zoonotic variables. Socioeconomic factors including the availability of serological test systems, the use and quality of the diagnostic tools and different activities in the medical community influencing the awareness of TBE do also correlate to the number of reported cases [11]. Several new TBE foci have recently been found or rediscovered in the Nordic countries, such as Bornholm in Denmark, southern Norway and several foci in the south of Sweden [12–15]. Models based on the known TBE endemicity and climatic factors (temperature and vapor pressure variables, both climate surface and satellite-derived) for predicting areas suitable for TBEV circulation have been developed [11,16,17]. These models not only confirm established endemic areas, but also predict several areas so far not known to be endemic for TBE as suitable for TBEV circulation in central, western and northern Europe. These data have led to a discussion of whether of not there are areas of TBE endemicity that have been overlooked. The reasons could be lack of data and a belief in many regions that the infection does not exist, leading to underdiagnosis of clinical TBE cases. We decided to perform a multicenter, retrospective follow-up of clinical cases of verified CNS-infection of presumed viral origin, but with unknown etiology, to investigate if TBE cases had in the past remained undiagnosed in the chosen areas.

2. Material Patients with meningitis or meningoencephalitis with verified pleocytosis in cerebrospinal fluid (CSF) and of unknown etiology were retrospectively identified in the files of virological departments or departments of infectious diseases. After giving his/her informed consent, the individual was included in the study if one or more serum and/or CSF samples had been obtained during the clinical course of in-

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fection. Table 1 shows the number of individuals included from each collaborating center during the specified time periods (total 233 individuals). The centers were chosen based on geographical location, covering non- or low-endemic regions in the vicinity of identified endemic regions and known not to look for TBE on a routine basis in the serological follow-up of patients with viral CNS-infection.

3. Methods The available serum and/or CSF samples were serologically screened at local virological departments using a commercial ELISA test system for detecting TBEV IgM and IgG (Immunozym IgM and IgG TBE ELISA, Immuno GmbH, Heidelberg). The assay was used according to the manufacturer’s instructions. When a sample was analyzed as positive or borderline, a serological data sheet was completed. All available samples obtained during the course of infection from that individual were examined and later sent to the Institute of Virology, University of Vienna, for confirmation. The samples were re-evaluated using an in-house ELISA for detection of TBEV IgM and IgG. The absorbance values were transformed into Vienna units (63–126 VIEU are considered as borderline and >127 VIEU as positive) [18]. A neutralization assay (NT) using live virus in cell culture with final antigen detection by ELISA was used for confirmation [19]. The NT results are shown as NT100 which refers to the reciprocal serum dilution that was able to suppress virus infection so no viral antigen could be detected, and NT50 which gave a 50% reduction of the antigen ELISA value. NT100 titers ≥10 and NT50 titers >10 were considered positive. For all patients found positive or borderline in the screening, a case–control questionnaire was completed based on medical records. If possible, an interview with the individual

Table 1 Involved centers (name and geographical coverage) and number of included individuals from specified time period Center

Region, country

No. of individuals included in the study

Time period

Department of Infectious Diseases, Sahlgrenska University Hospital, Östra sjukhuset, Gothenburg Department of Infectious Diseases, County Hospital, Kalmar Department of Infectious Diseases, Central Hospital, Kristianstad Department of Infectious Diseases, Central Hospital, Västerås

County of Västra Götaland, Sweden (west)

53

1997–2000

County of Kalmar, Sweden (south-east) County of Kristianstad, Sweden (south) County of Västmanland, Sweden (inland, west of Stockholm County) The Netherlands

32 29 6

1990–2000 1997–2000 1998–2000 1995–2000

Belgium Lorain, France

16 (all negative in screening) 4 70

2000 1999–2000

Besancon region, France

23

1999–2000

National Institute of Public Health and Environment (RIVM), Amsterdam Medical Microbiology, University of Li`ege Infectious and Tropical Disease Unit, Laboratoire de Virologie, Hopital de Brabois Adultes, Centre Hospitalier et Universitaire de Nancy, Vandoeuvre-l`es-Nancy Infectious Diseases and Tropical Medicin, Laboratory of Bacteriology, CHU de Besancon, Hopital Saint-Jacques, Besancon

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Table 2 Individuals found borderline or positive in the ELISA screening Patient no. and location of center

Sex/age

Noticed tick-bite within the last 4 weeks before illness

1/Nancy 2/Nancy 3/Nancy 4/Nancy 6/Nancy 7/Nancy 8/Nancy

F/62 F/21 F/38 M/30 M/49 F/33 M/48

No Vosge, France No No No No No

1/Besancon 2/Besancon 3/Besancon 4/Besancon 5/Besancon 6/Besancon 7/Besancon 8/Besancon 9/Besancon 10/Besancon 11/Besancon 12/Besancon

M/21 F/70 M/77 M/30 F/27 M/31 F/34 F/32 M/23 F/0 F/40 M/21

1/Li`ege 2/Li`ege 3/Li`ege 4/Li`ege

Active immunization

Clinical diagnosis

Clinical outcome

TBE

Yellow fever

Japanese encephalitis

No No No No No No No

No No No No No No No

No No Yes, 1990 Yes No Yes, 1995 Yes

No No No No No No No

Men-enc Men Men Men Enc-myel Men Enc-myel

Partial rec. Full rec. Partial rec. Full rec. Full rec. Full rec. Partial rec.

DM DM DM DM DM DM DM DM DM DM DM DM

DM DM DM DM DM DM DM DM DM DM DM DM

DM DM DM DM DM DM DM DM DM DM DM DM

DM DM DM DM DM DM DM DM DM DM DM DM

DM DM DM DM DM DM DM DM DM DM DM DM

Myel Men-enc Men Men Men Men Men Men Men-enc Men Men Men

Full rec. Unknown Partial rec. Full rec. Full rec. Full rec. Full rec. Full rec. Full rec. Full rec. Full rec. Full rec.

F/86 M/75 F/24 F/65

DM DM No No

DM DM No No

DM DM No No

DM DM DM DM

DM DM DM DM

DM Locked-in syndrome Radiculitis DM

DM Fatal Partial rec. Partial rec.

1/Västerås

M/86

DM

DM

DM

DM

DM

Men-enc

Partial rec.

1/Kristianstad 2/Kristianstad 3/Kristianstad

M/59 M/68 F/87

No Stockholm, Sweden No

DM Yes No

No No DM

DM No DM

DM No DM

Recurrent men Men Men

Full rec. Full rec. Full rec.

1/Kalmar

M/51

Kalmar, Sweden

No

No

No

No

Men-enc

Full rec.

1/Gothenburg 2/Gothenburg 3/Gothenburg 4/Gothenburg

M/25 M/26 M/31 M/27

No No DM DM

No No DM DM

No No DM DM

No Yes, 1975 DM DM

DM No DM DM

Men-enc Men DM DM

Full rec. Full rec. DM DM

Concomitant disorders

HIV-positive HIV-positive HIV-positive

Bierner dis.

Systemic inflammatory dis./SLE

No patient consumed unpasteurized milk before illness or received passive immunoprophylaxis after noticed tick-bite, F: female, M: male, DM: data missing, rec.: recovery, Men: meningitis, Men-enc: meningoencephalitis, Enc: encephalitis, Enc-myel: encephalomyelitis, Myel: myelitis, dis.: disease.

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Visit to known TBE endemic region within the last 4 weeks before illness

M. Haglund et al. / Vaccine 21 (2003) S1/66–S1/72

was performed. The questionnaire covered previous TBEvaccination or the use of TBEV specific immunoglobulins as post-exposure prophylaxis and vaccinations with crossreactive flaviviruses (yellow fever, Japanese encephalitis). For the verified cases of acute TBE we asked for preceding tick-bite and the probable geographical origin of the infection. A short comment concerning the clinical picture was also asked for.

4. Results Out of 233 included individuals, 32 were found either positive (n = 10), or borderline (n = 22) in the screening procedure (Tables 2 and 3). Twenty-four individuals were found positive and eight had only borderline results when adding the results from the in-house ELISA. A majority of the samples were positive or borderline only in the IgG ELISA. Two cases of acute TBE were identified from two different centers in Sweden (Kalmar, Kristianstad) and were confirmed using the in-house IgM and IgG ELISA and NT. The patient from Kristianstad (until 2001 considered a non-endemic area) was probably not infected in the county of Kristianstad. He had visited endemic areas in the Stockholm archipelago during the weeks preceding the disease where he also noticed a tick-bite. The patient with acute disease from Kalmar was a permanent resident in a village located at the Baltic Sea just north of the city of Kalmar. He had not left the Kalmar area in the weeks before he fell ill.

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One 87-year-old woman with known systemic inflammatory disease was found positive in the IgG ELISA and the NT indicating a specific TBEV reactivity (Table 2). She had no known history of a previous TBE, TBE vaccination or meningoencephalitis of unknown etiology. One 26-year-old man was also found IgG ELISA and NT positive. He had no history of previous TBE or TBE vaccination but had a yellow fever vaccination in 1975. One 86-year-old man was found IgG ELISA and NT positive in a late convalescent sample. No data available describing his medical history. All these five individuals were included from Swedish centers. No NT positive individuals were found in France, Belgium or The Netherlands. A known exposure to other flaviviruses (yellow fever vaccination) was identified in four individuals among samples found IgG reactive but IgM non-reactive in the ELISA test systems and without specific reactivity in NT. This indicates that cross-reactivity between these two flaviviruses was a reasonable explanation for the ELISA IgG reactivity in these specific samples. A comparison between the results from the two IgG TBE ELISAs indicates that the “Vienna” IgG ELISA results in a higher proportion of IgG reactive samples compared to the Immunozym IgG kit (Table 3). The correlation between the two IgM TBE ELISA test systems was satisfactory (Table 3). None of the patients had knowingly consumed unpasteurized milk before the illness and none of them had received passive immunoprophylaxis after tick exposure (0/16, no information available for the remaining 16 individuals).

Table 3 Serological results for individuals found borderline or positive in ELISA screening Patient no. and location of center

Date of sampling

ELISA screening

Reference laboratory, Institute of Virology, University of Vienna

IgM

IgG

ELISA IgM

ELISA IgG

NT 100

NT 50

1/Nancy

990514 990527 990623

Bl Bl Neg

Neg Bl Bl

Neg Neg Neg

Neg Neg Neg

Neg Neg Neg

Neg Neg Neg

2/Nancy 3/Nancy

990707 990809 000309 000412

Neg Neg Bl Neg

Bl Neg Bl Neg

Neg Neg Neg Neg

Bl 224 169 145

Neg Neg Neg Neg

Neg Neg Neg Neg

4/Nancy

991004 991005 991108 991115 991122

Neg Neg Neg Neg Neg

Bl Neg Bl Bl Neg

Neg Neg Neg Neg Neg

278 258 288 309 287

Neg Neg Neg Neg Neg

Neg Neg Neg Neg Neg

6/Nancy

000113 000121 000127 000201 000209 000309

Neg Neg Neg Neg Neg Neg

Neg Neg Neg Neg Neg Bl

Neg Neg Neg Neg Neg Neg

Neg Neg Neg Neg Neg Neg

Neg Neg Neg Neg Neg Neg

Neg Neg Neg Neg Neg Neg

7/Nancy

000208 000307

Neg Neg

Bl Bl

Neg Neg

137 180

Neg Neg

Neg Neg

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Table 3 (Continued ) Patient no. and location of center

Date of sampling

ELISA screening

Reference laboratory, Institute of Virology, University of Vienna

IgM

IgG

ELISA IgM

ELISA IgG

NT 100

NT 50

8/Nancy

000309 000310 000311 000323 000328

Neg Neg Neg Neg Neg

Neg Bl Neg Neg Neg

Neg Neg Neg Neg Neg

>1000 936 881 >1000 >1000

Neg Neg Neg Neg Neg

Neg Neg Neg Neg Neg

1/Besancon 2/Besancon 3/Besancon

990127 991015 990922 000222

Neg Neg Neg Neg

Bl Bl 230 170

Neg Neg Neg Neg

196 153 567 558

Neg Neg Neg Neg

Neg Neg Neg Neg

4/Besancon 5/Besancon 6/Besancon

000512 000630 000605 000622

Neg Neg Neg Neg

Bl Bl Bl Bl

Neg Neg Neg Neg

147 176 301 264

Neg Neg Neg Neg

Neg Neg Neg Neg

7/Besancon 8/Besancon 9/Besancon 10/Besancon 11/Besancon 12/Besancon

000902 000529 000107 000218 000801 (conval.) 001009

Neg Neg Neg Neg Neg Neg

Bl Bl Bl >350 Bl Bl

Neg Neg Neg Neg Neg Neg

Neg 203 164 >1000 Neg Neg

Neg Neg Neg Neg Neg Neg

Neg Neg Neg Neg Neg Neg

1/Li`ege 2/Li`ege 3/Li`ege 4/Li`ege

000626 000629 000720 000720

Neg Bl Neg Neg

Bl Neg Pos Bl

Neg Neg Neg Neg

Bl Neg 275 128

Neg Neg Neg Neg

Neg Neg Neg Neg

1/Västerås

0006xx (conval.)

Neg

Bl

Neg

306

10

20

1/Kristianstad

970108 (acute) 000906 (acute)

Neg Neg

Bl 145

Neg Neg

428 381

Neg Neg

Neg Neg

2/Kristianstad 3/Kristianstad

000906 000724

510 Neg

>300 Bl

568 Neg

>1000 766

30 40

160 40

1/Kalmar

910930 911002

650 825

175 300

>1000 >1000

>1000 >1000

Neg 15

80 240

1/Gothenburg

970516 970516 (csf)

Neg Neg

320 180

Neg Neg

>1000 113 (Bl)

Neg Neg

Neg Neg

2/Gothenburg

980929 981013

Neg Neg

210 132

Neg Neg

>1000 >1000

20 30

30 120

3/Gothenburg 4/Gothenburg

970120 981012

Neg Neg

140 180

Neg Neg

235 626

Neg Neg

Neg Neg

All available samples include, Bl: borderline, Neg: negative, Conval.: convalescent.

5. Discussion Recently, the true endemicity of TBE in Europe has been discussed. Valid data describing both a decrease and an increase of TBE endemicity are reported [1,7,8,10,13,15,20]. There are several areas in Europe not known to be TBE endemic that have been considered suitable for circulation of TBEV based on climatic and vegetational data [11,16,17]. In most regions considered non-endemic, TBE is not screened for on a routine basis in the clinical and serological evaluation of patients with viral CNS-infection. Even in some areas known to be endemic, especially those being low-endemic, serological screening for TBE is not performed as a routine procedure. These data indicate the existence of undiscov-

ered areas of TBE endemicity in Europe and suggest the possibility of undiagnosed TBE cases in several regions. Our retrospective serological evaluation of presumed viral CNS-infections without established etiology revealed only two previously undiagnosed cases of acute TBEV infection. One patient had probably been infected in the Stockholm area in Sweden, a known TBE endemic focus. The second patient had been infected in the county of Kalmar, a known low-endemic region in the southeast of Sweden. The number of previously undiagnosed cases of TBE was somewhat lower than expected, but so was the number of samples provided by some centers. In addition, the departments participating in the present study could cover only limited geographical areas and we could not identify any TBE cases

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from France, Belgium or The Netherlands. However, there are still several regions in Europe where undetected endemic foci may be identified by serological screenings, for example parts of Germany, Switzerland, eastern France, northern Italy and southern parts of both Sweden and Norway. Examples of rediscovered areas with human TBE during recent years are the island of Bornholm (Denmark) and southern Norway [13,15,21]. In both areas investigations were performed during the 1960s and 1970s proving the circulation of TBEV, or a TBEV-like virus. These data did not result in serological screening for TBE in clinical practice [22,23]. In recent years, TBE cases have been confirmed in both areas and there is reason to believe that during the past decades TBE cases have remained undiagnosed due to the low awareness among local physicians. In most European countries, including Sweden, an increase in reported annual cases of TBE has been found over the past two decades. The endemic areas in Sweden were considered stable [24]. TBE endemic areas are located in the east, mainly in the counties of Stockholm, Uppsala and Södermanland. Most of the foci are located in the vicinity of Lake Mälaren and the Baltic Sea [12–14,24]. Outside these areas only a smaller proportion of patients with viral CNS-infection has been serologically examined for TBEV. In recent years, several new TBE-foci have been found outside the established endemic areas. Approximately 50 human cases of TBE are reported from these foci. These so-called new foci are found mainly in the Skåne and Blekinge region in the far south of the country and in several locations around the two big lakes Vättern and Vänern [12–14,25]. Both lakes are located in the central parts of Sweden. The reasons for this changing epidemiological situation in Sweden are partly unknown at present. Some influential factors have been evaluated. An increase in density and a spread to the north have been reported for both ticks and roedeer (an important amplifying host for ticks). A correlation between milder climate and the number of human TBE cases has been shown [26–28]. These foci may indicate the establishment of real new foci of TBEV circulation caused by the introduction of TBEV to new suitable habitats. Another possibility is the previously low awareness of TBE, resulting in undiagnosed TBE cases. The Swedish results from this study produced more valid data only from three regions (Gothenburgh, Kalmar and Kristianstad) indicating that there have been few undiagnosed cases of TBE in the past in these particular areas. Interestingly, during the years 2001 and 2002, four cases of TBE were diagnosed at the Department of Infectious Diseases in Kristianstad, all infected in the same area northwest of Kristianstad. Another issue is the true number of TBE patients infected during travel to endemic areas abroad. These patients can fall ill after they return back home and may be hospitalized in areas where the awareness of TBE is low. Because of this low awareness, these TBEV infections may remain undiagnosed [1,25,29].

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Another important issue in the field of flaviviral diagnostics is the risk of cross-reactivity between different flaviviruses when ELISA assays are applied [19,30,31]. The same is true for hemagglutination inhibition and complement binding assays, even though they are more rarely used nowadays in routine diagnostics. The cross-reactivity seen in ELISA can be a problem in the diagnosis of acute TBE in an individual previously exposed to another flavivirus (includes both natural infection and vaccination), in seroprevalence studies or when TBEV-immunity is evaluated. In the latter two cases, there is also the possibility of a non-specific, often low, IgG-reactivity that cannot be confirmed in NT in an individual free from flavivirus exposure [32,33]. The phenomenon of cross-reactivity presented in this study as an ELISA IgG-reactivity in individuals with a history of yellow fever vaccination. This emphasizes the importance of anamnestic data, including medical history and vaccination status, in individuals where TBEV serology is used for one or the other reason. To conclude, few cases of undiagnosed acute TBE were found in this limited study comprising eight centers in four countries. The available data describing TBE epidemiology suggest that new areas of TBE endemicity are yet to be found. A certain underdiagnosis of individual TBE cases related to missing data and a low awareness in the medical community exists. Our data confirmed the importance of an adequate medical and vaccination history in a patient where serology for TBEV, or other flaviviruses, is to be performed in order to avoid the risk of misinterpretation of positive results caused by crossreactivity.

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