Tick-borne encephalitis among patients with viral meningitis in Bulgaria

Considering the remarkable increase in TBE morbidity in Europe over the past two decades [28], we organized and conducted a study of TBE among patients with acute viral meningitis, who were hospitalized in Bulgaria during 2009-2012.

A total of 86 patients with acute viral meningitis were investigated between 2009 and 2012 by physicians at the infectious diseases units at regional hospitals in districts of Sofia, Pazardzhik, Plovdiv, and Burgas. A total of 86 serum samples were collected during the acute phase and 49 sera at the convalescence phase up to 30 days after the first sample.

All 135 serum samples from patients were tested for IgM antibodies, and positive were also tested for IgG antibodies against TBE virus using commercially available ELISA tests (Euroimmun, Germany), according to the manufacturer's instructions.

TBE virus RNA was detected by reverse transcription polymerase chain reaction based on quantitative real-time technology (TaqMan) as described [29]. The system detected a fragment of the 3' noncoding region of the TBE virus genome.

A total of 86 patients with viral meningitis of unknown etiology during this period were tested to detect acute TBE. Three TBE cases in Bulgaria were found. The last TBE case was detected in October 2012 and the other two were diagnosed in 2009.

3.3.1 Case no. 1

A girl aged 16 years residing in Velingrad (South Bulgaria) was admitted to the regional hospital on April 10, 2009. The patient had a high fever (40°C) and malaise. The temperature went to normal 3-4 days after admission, and then again her condition deteriorated with fever, headache, stiff neck, sore throat, nausea, vomiting, and depressed mood. The patient had a history spending some time in the forest. The cerebrospinal fluid (CSF) collected on April 14 showed a high number of leucocytes (160∕μL; norm, 0-5∕μL) with 75% granulocytes, high protein content (125 mg∕dL, norm 15-45 mg∕dL), and normal glucose level (0.31 mmol/L; norm, 0.22-0.44 mmol/L).

3.3.2 Case no. 2

On September 11, 2009, a 21-year-old man was admitted to the regional hos­pital in Plovdiv (South Bulgaria) with fever (38.5°C), fatigue, headache, nausea, and vomiting. Stiff neck, stupor, muscle soreness, conjunctivitis, and abnormal reflexes with pain in joints were found during physical examination. The onset of the disease was 5-6 days earlier. Exposure to tick bites could be excluded. The CSF analysis showed increased count of leucocytes 301∕μL (norm: 0-5∕μL) with 82% lymphocytes, slightly elevated protein (56 mg∕dL; norm, 15-45 mg∕dL), and normal glucose level (0.38 mmol∕L; norm, 0.22-0.44 mmol∕L). The patient initially improved and after a week, the patient’s condition worsened again. He manifested fever, significant dizziness, and severe headache. The CSF analysis also supported worsening of the patient. Leucocyte count reached 442∕μL (norm: 0-5∕μL), with 90% lymphocytes, and protein was remarkably elevated (134 mg∕dL; norm, 15-45 mg∕dL); glucose level (0.28 mmol∕L, norm, 0.22-0.44 mmol∕L) was normal. Within a month, the patient gradually recovered. Examination by ELISA of paired serum samples from the patient, one upon admission and a second during the reconvalescence, revealed high level of IgM antibodies and no IgG antibodies in the first serum sample and borderline level of IgM antibodies in the first sample and significant levels of IgG antibodies against TBEV in the second serum sample [26].

3.3.3 Case no. 3

A 28-year-old woman, resident of Burgas area (East Bulgaria), was admitted to the regional hospital on September 23, 2012, with fever (37.5-38⁰ C), significant numbness in muscles, and weakness. Physical examination revealed mild neck stiffness, mild left hemiparesis, and distal-type hypoesthesia. Her medical history started 2 days before. Upon admission, a tick was found on her body and removed. On September 27 the patient’s condition improved, but starting from October 1, the fever, weakness, and numbness in muscles exacerbated. CSF analysis showed slightly elevated leukocytes (60∕μL; norm, 0-5∕μL) and protein (74 mg∕dL; norm, 15-45 mg∕dL), normal glucose level (0.38 mmol∕L; norm, 0.22-0.44 mmol∕L). Two serum samples, taken on October 1 and October 10 were tested by ELISA, and both antibodies, IgM and IgG, specific to TBE virus were detected. The patient was discharged in improved condition.

The serum samples of the three patients tested negative by ELISA and IFA for West Nile and yellow fever viruses, also negative for IgM antibodies to Borrelia burgdorferi by ELISA. Their CSF samples tested negative for bacterial culture. Though TBE cases are reported sporadically, TBE virus circulates in the country, causing human cases associated either with tick bites or consumption of unpasteurized milk.

In all three patients described, typical biphasic course of TBE infection was revealed. About two-thirds of the patients develop only febrile syndrome in the first phase of the disease [30]. Neurological disorders appear during the second febrile phase. Biphasic febrile illness is typical for infection with Western subtype of the virus. Patients infected with Eastern subtype of TBEV develop only monophasic course [30].

TBE cases in humans are occasionally reported in Bulgaria. However, the fact that TBE cases occur in Bulgaria, even sporadically, and are associated with tick bites or consumption of unpasteurized milk shows that TBE virus circulates in the country.

There is significant increase in the number of registered cases of TBE in Europe, Russia, and Far East, starting with 1990 [28]. Since then, about 10,000-12,000 TBE cases are reported annually in Europe and Russia. There is a tendency to global increase in the number of cases and to expansion of areas at risk. In Sweden, a significant increase in TBE cases reported was recorded in the last decade [31]. New endemic areas in Switzerland were confirmed by detection of TBE virus RNA in field-collected ticks [32]. Since September 2012, considering the importance and spread of TBE in the European Union, European Commission included TBE in the list of communicable diseases covered by epidemiological surveillance in the member states [33].

The three cases reported considered the first clinically and laboratory confirmed cases in Bulgaria since. The first case proved to have mixed infection with M. tubercu­losis that could promote the primary progressive course of the meningoencephalitis, as previously reported [34]. The second case showed clinical manifestation of subacute viral meningitis, while the third case presented as subacute encephalomyelitis.

Usually, IgM and IgG antibodies to TBEV are present by the time that central nervous system involvement manifests in the second stage of TBE. Nucleic acids of the TBEV are very rarely detected by PCR during the viremic stage of the disease [29]. Surprisingly, we detected TBE virus infection by RT-PCR in the first patient. Thus, we confirmed not only the case but also the real circulation of the virus in Southeast Europe, where no information is available so far. The first case described above was also remarkable by the two coinfections ongoing—TBE and tuberculosis, responsible for aggravation of the course of the illness.

The TBE cases described showed that the disease is probably not uncommon in Bulgaria. The risk of TBE is underestimated in Bulgaria because of the low aware­ness of medical doctors. TBE should be taken into consideration in patients with various manifestations of central nervous system infections in Bulgaria.

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