Introduction

Tick-borne encephalitis virus (TBEV) is a positive strand RNA virus in the genus flavivirus usually transmitted to humans by Ixodes ricinus ticks, or less frequently by consumption of dairy products from infected domestic animals [1].TBEV infection often occurs in a biphasic manner [2]. The first viremic phase is characterised by flu-like symptoms, which after a shorter period with few symptoms, may be followed by a second phase affecting the central nervous system with symptoms of meningitis and/or encephalitis.

The diagnosis of tick-borne encephalitis (TBE) often relies on the presence of TBEV-specific IgM alone or both IgM and IgG antibodies in serum, as the clinical manifestations are not specific for this disease [3, 4]. The serological tests may be precluded by IgM antibodies induced by recent TBE vaccination or long-lasting IgM from previous asymptomatic or mild infection of TBEV. False positive test results may also arise from cross-reactive antibodies remaining from infection by or vaccination against other flaviviruses such as dengue, Zika, yellow fever or Japanese encephalitis viruses [5, 6]. Testing for TBEV neutralising antibodies in serum will verify the presences of TBEV antibodies, yet antibodies induced by TBE vaccination cannot be excluded. Verification of an uncertain serology requires either detection of a four-fold increase in TBEV antibody titre in serum samples taken a least two weeks apart or TBEV antibodies produced in the cerebrospinal fluid (CSF) determined by a positive CSF/serum antibody index [7, 8]. Due to more travelling and vaccination, antibodies cross-reactivity in TBEV serology tests are rising in the population. Thereby, alternative approaches for diagnosing TBE could be beneficial to circumvent the difficulties with antibody testing.

In the first viremic phase of the disease, TBEV viral RNA present in blood may be detected by molecular methods. In the neurologic phase, when most patients seek medical care due to severe symptoms, TBEV RNA is rarely detected neither in blood nor in CSF [9]. A few cases have been reported where TBEV has been detected in urine from patients in the neurologic phase [10–12]. Persistent viremia and urine shedding of TBEV have been demonstrated for more than six weeks in an immunosuppressed patient with persistent infection [11]. Other flaviviruses have also been detected in patient saliva [13].

The aim of this study was to evaluate if detection of TBEV RNA could be useful in diagnosing TBE in the neurologic phase. Samples of blood, urine and saliva from immunocompetent Norwegian patients hospitalised with suspected aseptic meningitis/encephalitis, were collected and tested by PCR.

Material and methods

Reverse transcription and real-time PCR assays

PCR analysis at Sørlandet Hospital.

RNA (5 μl each) from saliva, urine and EDTA-blood was reverse transcribed to cDNA by using the qScript^TM cDNA synthesis kit (Quanta BioScience Incorporated, Gaithersburg, MD, USA). Samples (5 μl cDNA) were tested by two real-time Taqman PCR protocols (designated TBEV1 and TBEV2, Table 1) [14, 15]. The TBEV1 and TBEV2 PCR amplifies a 69 base pair (bp) in the 3’ non-coding region and 54 bp fragment located on the envelope gene of TBEV, respectively. Protocols were performed using 5μl of cDNA in a 15 μl reaction mixture consisting of 5mM MgCl_2, 0.5 units Uracil DNA-glycosylase (Eurogentec S.A. Seraing, Belgium), LightCycler FastStart DNA master mix (Roche, Mannheim, Germany) with the primer and probe concentrations given in Table 1 and run on a LightCycler 480 with the following thermocycling parameters; 2 min at 40°C followed by 10 min at 95°C and 45 cycles of 15 s at 94°C, 45 s at 60°C and 30 s at 72°C.

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Table 1. Characteristics and sequences of TBEV-PCR primers and probes used in in this study.

https://doi.org/10.1371/journal.pone.0305603.t001

PCR analysis at Norwegian Institute of Public Health.

RNA (5 μl) were reverse transcribed to cDNA by High High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster city, CA, USA). Samples (3 μl cDNA) were analysed by TBEV2 PCR (Table 1) performed according to Andreassen et al. [15].

Results

Serology

A total of 31 hospitalised patients suspected to have viral infection of the central nervous system were included in the study, and 11 of them were classified as TBE cases primarily based on TBEV antibody testing in serum. All TBE cases had detectable TBEV-specific IgM and IgG antibodies (Table 2). Three patients were tested in consecutive sera, and TBE was confirmed by either increase in both TBEV IgM and IgG or increase in TBEV IgG and decrease in IgM levels. The 20 patients classified as non-TBE, except for two with detectable serum TBEV IgG only, had no detectable TBEV antibodies.

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Table 2. Presence of serum TBEV IgG/IgM antibodies and characteristics of TBE patients.

https://doi.org/10.1371/journal.pone.0305603.t002

Discussion

TBEV RNA was not detected by RT-PCR in neither serum, whole blood, saliva nor urine from any of the 11 immunocompetent TBE patients in the second neurologic phase, hospitalised at Sørlandet Hospital, Norway. Concentrated saliva (2x) and urine (4x) samples were analysed to increase the rate of TBEV RNA detection without any success. All TBE patients had both detectable virus-specific IgM and IgG at the time patient samples were analysed by two different TBEV RT-PCR analyses, indicating that most if not all patients were late in the disease course.

Our findings contrast with what reported previously for four TBE patients in the neurologic phase, where TBEV RNA was detected in urine samples [10–12]. Three of the patients presented in these studies were on immunosuppressive drugs and had severe disease courses. One of them had detectable IgG in serum and TBEV RNA in urine at day 49 after disease onset, another had detectable IgM only and TBEV RNA present in urine from day 7–16 and the third had no detectable TBEV antibodies whereas TBEV RNA were present in both CSF and urine at the day of hospitalisation [10–12]. An immunocompetent patient seroconverted during the hospital stay (day 17) and TBEV RNA was detected in urine at day 19 [10]. No TBEV RNA was detected in this patient four days later. It should though be underlined that detection of TBEV RNA in urine is a sporadic event and in most cases the samples are negative [16].

The TBE patients in our study were immunocompetent and late in their disease course thus they all had detectable virus-specific antibodies. Antibodies are significant for viral clearance, as demonstrated by others TBEV RNA in blood is disappearing when TBEV IgM and IgG antibodies are appearing [17]. Along this line, the presence of TBEV-specific IgM is reported to be associated with a mild disease presentation [18] whereas long lasting low levels of virus-specific IgG are associated with severe and prolonged clinical courses [19]. All but two of the TBE patients in our study had hospital stays for nine days or less, consistent with a mild/moderate illness probably due to a well-established immunity against the infection.

A serum sample from one of the patient in our study, with suspicion of mononucleosis 25 days prior to the samples collected for our study, had at that time detectable levels of TBEV RNA. At the time, the patient presented with fever and sore throat. This is in agreement with the notion that TBEV are occurring in the bloodstream in the early phase of infection.

CSF samples were not collected from our study patients. Avoiding an unnecessary invasive procedure was likely to increase the chance that most of the few TBE patients admitted to the hospital consented to participate. Using molecular methods on CSF has been reported to have low diagnostic sensitivity in TBE in other studies [20, 21]. It should, however, be emphasized that molecular detection of viral RNA in CSF may have diagnostic value in certain patient cases, e.g. when patients have not developed neutralising antibodies, are immunosuppressed and/or if the disease is severe and progressive.

Our data suggest that molecular detection of TBEV RNA in blood and body fluids as urine and saliva will mostly not be of any diagnostic value in TBE patients with neurological complications and a moderate course of disease where serum IgM or IgG already is established. Uncertain cases may nevertheless be confirmed by antibody testing, either by observing an increase in antibody titre in paired sera or detection of intrathecal TBEV IgM. Further research is required to clarify the utility of TBEV RNA detection for the diagnostics and monitoring of TBE in particular disease cases, especially for those patients that are negative for IgM and IgG serology with clinical symptoms of viral infections. Viral RNA is useful in surveillance and phylogenetic studies, though not for diagnostic purposes in later stages of disease, at least in immunocompetent patients.

Acknowledgments

We are grateful for all help and support by colleagues at Department of Medical Microbiology, Sørlandet Hospital.