Results of the Screening of Tick-Borne Encephalitis Virus Antibodies in Human Sera from Eight Districts Collected Two Decades Apart

Abstract

The sera from healthy individuals aged 10–59 years randomly selected from the general population during repeated cross-sectional surveys were stored at −20°C at the serum bank of the National Institute of Public Health in Prague. The sera included in the present study were collected in the 1980s and in 2001 in eight districts of the Czech Republic. The proportional representation of the study localities was similar in both periods. The sera were uniformly distributed in 5-year age groups for 10- to 19-year-olds and in 10-year age groups for 20- to 59-year-olds. Males and females were nearly equally represented in both periods. Altogether 704 sera, 434 from the period 1978–1989 and 270 from 2001, were screened for antibodies against tick-borne encephalitis virus (TBEV) by the virus neutralization test. The seroprevalence rates were 11.5% in the 1980s and 26.3% in 2001. From the logistic regression model, it follows that the chance of detecting anti-TBEV antibodies was more than twice higher in 2001 than in 1978–1989 (odds ratio [OR]=2.22). The differences between males and females were not statistically significant, nor was sex–period interaction. The time trends in the seropositivity rates were similar in all age groups, with the exception of the 10- to 14-year-olds (p=0.914). The rate of seropositives in the age group 15–59 years increased 1.9 times, whereas that in the age group 10–14 years rose 5.1 times. In areas comparable to those where the study sera were collected, the average incidence rates of tick-borne encephalitis (TBE) per 100,000 population aged 10–59 increased significantly from 3.35 in 1978–1989 to 8.96 in 2001 (p<0.001). The age-specific antibody trends in adult age groups in both periods suggest that clinically manifest or inapparent TBE cases do not induce lifelong immunity, but they are likely to reflect the previous epidemiological situation.

Introduction

In Europe, the tick-borne encephalitis virus (TBEV) was first isolated from human cases and Ixodes ricinus ticks in the Czech lands (Bohemia and Moravia) of the former Czechoslovakia in 1948 and 1949 (Krejčí 1949, Gallia et al. 1949, Rampas and Gallia 1949. Krejčí 1950). Data on cases of tick-borne encephalitis (TBE) in the Czech lands have been available since the 1960s. TBE morbidity showed a slightly downward trend from 1970 to the late 1980s, with the annual rates ranging from 1.3/100,000 to 5.8/100,000 population. Since the early 1990s, an upward trend has been noted, with the annual rates ranging from 1.3/100,000 to 10.0/100,000 population.

In the present study, we compare the anti-TBEV antibody detection rates in human sera collected two decades apart, i.e., mostly in the 1980s (from 1978 to 1989), and in 2001 from the identical districts in both time periods.

Material and Methods

Collection of blood samples from healthy individuals for the multipurpose serological surveys started in the Czech Republic in year 1964. The blood samples were collected at random in preselected districts and estimated amounts in all age groups every year during autumn months. After centrifugation, the sera were separated in aliquots of 0.3 mL in sealed ampules. No names of donors were recorded. The documentation of sera included year of collection, age, sex, and vaccination (if available only). No information regarding the TBE vaccination was available. The sera were stored frozen at −20°C in the serum bank of the National Institute of Public Health in Prague. The last serological survey was conducted in year 2001. The sera selected for this present study were collected in the same regions in both time periods. The persons included in collections of sera during the 1980s were not vaccinated against TBE. The TBE vaccine was not generally available in the Czech Republic at that time. The voluntary vaccination campaign started in the 1990s and was targeted at schoolchildren. However, no central register of vaccination exists in the Czech Republic, therefore information regarding the vaccinations status of persons is not available. According to the reports from the vaccine production firms, the overall vaccination coverage at the beginning of new century did not exceed 15% of the population, with no information regarding the number of doses administered and their intervals.

The sera were collected in eight districts of six administrative regions (NUTS 3) of the Czech Republic (i.e., CZ020 Central Bohemian Region, CZ031 South Bohemian Region, CZ032 Plzeň Region, CZ064 South Moravian Region, CZ063 Highlands, and CZ080 Moravian-Silesian Region) in two time periods (1978–1989 and 2001). The proportional representation of the study localities was similar in both periods. The sera were uniformly distributed in 5-year age groups for 10- to 19-year-olds and in 10-year age groups for 20- to 59-year-olds. Males and females were nearly equally represented in both periods. Altogether 704 sera, 434 from the period 1978–1989 and 270 from 2001, were screened for antibodies against TBEV, Borrelia burgdorferi sensu lato (s.l.), and Anaplasma phagocytophilum. The results of the screening of antibodies against Lyme borreliosis and anaplasmosis will be presented in a separate paper.

Sera were investigated using the plaque reduction neutralization microtest (PRNT) on VERO E6 cells in flat-bottomed, 96-well, sterile microplates for tissue culture with covers (Sarstedt) and TBEV strain Hypr, a prototype of the Central European encephalitis subtype of TBEV (Sikutova et al. 2009). Briefly, 30 μL of thermally inactivated (at 56°C for 30 min) serum samples diluted 1:10 (screening) in Leibowitz L-15 medium with antibiotics were mixed with 30 μL of a test dose of the virus (30–50 plaque-forming units [pfu]) in L-15 medium with 3% fetal calf serum (FCS; Sigma) for cell culture. The serum–virus mixture was incubated at 37°C for 60 min. A total of 60 μL of Vero E6 cells (grown at 37°C for 3–4 days) suspension in L-15 with 3% FCS and antibiotics was added to each test well at a concentration about 20,000 cells per well. After incubation at 37°C for 4 h, 120 μL of overlay (1.5% carboxymethylcellulose sodium salt in L-15 supplemented with 3% FCS and antibiotics) was added to each well. The microplates, covered with lids and sealed in small plastic bags, were incubated at 37°C. The cell cultures were checked for plaques and cytopathic effect under an inverse microscope after 3 and 4 days and stained with 0.1% Naphthalene Black. Serum samples that revealed 90% or greater reduction of pfu numbers at the 1:10 dilution were considered positive.

Data from the EPIDAT, the Czech reporting system of notifiable diseases (National Institute of Public Health 2014) and reports of the Institute of Health Information and Statistics (Institute of Health Information and Statistics 1978–2001) were used to calculate the incidence of TBE per 100,000 permanent residential population. The results are presented as absolute and relative frequencies. The Fisher exact test and the multiple logistic regression model were used for the comparison between the periods and groups. All tests were evaluated at a significance level of 0.05. The data were analyzed by the software package Stata (release 9.2, Stata Corporation, College Station, TX).

Results

TBEV neutralizing antibodies were detected in 50 (11.5%) of 434 sera from 1978–1989 and in 71 (26.3%) of 270 sera from 2001 (Table 1). The rate of positive sera was clearly and significantly higher in 2001 than in 1978–1989 (p<0.001). Both males and females showed a similar trend in anti-TBEV antibodies in the study periods (Table 1). A statistically significant increase in the detection rates of anti-TBEV antibodies, from 13.4% to 25.5% (p=0.006) in males and from 9.8% to 27.1% (p<0.001) in females, was observed from the 1980s to 2001.

Table 1.

Detection of Anti-TBEV Antibodies in Sera by Sex and Period

	Period
	1978–1989		2001
Sex	No. of sera	Anti-TBEV antibody positivity	No. of sera	Anti-TBEV antibody positivity
Males	209	28 (13.4 %)	137	35 (25.5 %)
Females	225	22 (9.8 %)	133	36 (27.1 %)
Total	434	50 (11.5 %)	270	71 (26.3 %)

TBEV, tick-borne encephalitis virus.

From the calculation based on the logistic regression model, it follows that the chance of detecting anti-TBEV antibodies was more than two times higher in 2001 than in 1978–1989 (odds ratio [OR]=2.22). The differences between males and females were not statistically significant, nor was and sex–period either, which means that the female–male relationships did not change considerably between the periods in terms of seropositivity. These calculations were adjusted for age to control for potential difference in age distribution between the periods (Table 2).

Table 2.

Difference Between Females and Males and Sex–Period Interaction Analyzed by Logistic Regression, Adjusted for Age Group

Factor	Odds ratio	95% CI	p value
Period (2001 vs. 1978–1989)	2.22	1.27–3.89	0.005
Sex (females vs. males)	0.68	0.38–1.24	0.213
Sex–period interaction	1.59	0.71–3.60	0.260

CI, confidence interval.

Table 3 shows age distribution of the screening results by 10-year or 5-year (for10- to 19-year-olds) age groups. For the period 1978–1989, the lowest rates of positive sera were found in 10- to 14-year-olds (9.3%) and 30- to 39-year-olds (6.9%). The positivity rates in other age groups were nearly identical. In 2001, the lowest positivity rate was seen in 30- to 39-year-olds again (9.3%). In contrast, the highest rate (47.6%) of seropositivity was revealed in children 10–14 years of age. The rates of seropositivity in the other age groups ranged slightly from 23.3% to 27.1% (Table 3).

Table 3.

Age Distribution of TBEV Neutralizing Antibody Positives

	Period
	1978–1989		2001
Age group	No. of sera	No. (%) of positives	No. of sera	No. (%) of positives
10–14	75	7 (9.3%)	42	20 (47.6%)
15–19	74	10 (13.5%)	53	14 (26.4%)
20–29	70	8 (11.4%)	48	13 (27.1%)
30–39	72	5 (6.9%)	43	4 (9.3%)
40–49	70	9 (12.9%)	41	10 (24.4%)
50–59	73	11 (15.1%)	43	10 (23.3%)
Total	434	50 (11.5%)	270	71 (26.3%)

TBEV, tick-borne encephalitis virus.

Similar time trends in the seropositivity rates were observed in all age groups, with the exception of the 10- to 14-year-olds (p=0.914). The seropositivity rate in the age group 15–59 years increased from 12.0% in 1978–1989 to 22.4% in 2001, i.e., 1.9 times, whereas in the age group 10–14 years, it rose 5.1 times.

In areas comparable to those in which the study sera were collected, the average incidence rates of TBE per 100,000 population aged 10–59 increased significantly from 3.35 in 1978–1989 to 8.96 in 2001 (p<0.001).

Distribution of TBE age-specific (10–59 years) incidence/100,000 inhabitants and TBE antibody positivity in sera collected from healthy individuals aged 10–59 years according to the time period and region is presented in Table 4. From the table, it follows the TBE age-specific incidence in the period 1978–1989 differs accidently in all six regions. In all of them, the incidence increased in 2001. The TBE serum antibody positivity increased in five of them. The last one (Moravian–Silesian region) is the heavy metal industrial area with a high level of air pollution that influences the local natural ecosystems and the health status of the population as well (Table 4).

Table 4.

Distribution of TBE Age-Specific (10–59 years) Incidence/100,000 Inhabitants and TBEV Antibody Positivity in Sera Collected from Healthy Individuals Aged 10–59 Years According to the Time Period and Region

	TBE age specific incidence/100,000 inhabitants		Anti-TBEV antibody positivity (%)
	Period		Period
Administrative regions NUTS 3	1978–1989	2001	1978–1989	2001
Central Bohemian CZ020	0.1	6.0	25.0	26.5
South Bohemian CZ031	5.4	29.6	9.7	39.2
Plzeň CZ032	9.7	12.7	8.3	23.9
Highlands CZ063	1.4	9.8	14.6	21.2
South Moravian CZ064	4.0	4.8	8.5	22.9
Moravian-Silesian CZ080	1.2	3.0	7.4	3.6

TBEm tick-borne encephalitis; TBEV, tick-borne encephalitis virus.

Discussion

The selection of the study sera from the serum bank was limited by the number of the stored sera collected in the study areas during the study periods (Kriz et al. 2003). A certain disproportion in numbers of sera collected in both periods did not affect the validity of the study because the two groups of sera were comparable in terms of the distribution of age, sex, and region of residence. Although the population sample studied is not representative of the whole Czech Republic, it covers both low-incidence and high-incidence areas. The reason for selecting the period 1978–1989 was that no TBE vaccine was available in the 1980s in the Czech Republic and, therefore, the anti-TBEV antibodies detected only reflect the immunity induced by the wild TBEV. When becoming available in the following years, the TBE vaccine was not included in the childhood immunization schedule covered by insurance either, and the vaccine coverage rate remained very low. Optional vaccination was only provided in childhood age groups. In 2001, in the age group 10–14 years, the vaccine coverage rate in the study areas was about 22% (Cabrnochova et al. 2010).

TBE vaccination data of the persons selected for donation of blood samples for multipurpose serological surveys were not available. It can be assumed that the increased seropositivity rate in this age group was due to the contribution of targeted vaccination campaigns. Increasing interest of the childhood population in outdoor activities may also have played a role. After the change of the previous communist regime in 1990s, scout camp activities started that were previously suppressed. Quite primitive summer camps with tents of sleeping bags only become popular. Moreover, “green” activities, a return to nature way of living for young persons, were in favor as well.

The trend of age-specific TBE incidence has changed. An initially flat trend curve became two peaked. The first maximum was in the approximate age group 10–14 and the second one in older age group 50–64 (Daniel et al. 2011, Heinz et al. 2013). The Czech reporting system has been notifying clinically symptomatic laboratory-confirmed TBE cases since the 1970s and recently has followed the European Centre for Disease Prevention and Control (ECDC) case definition. Sensitivity and specificity of the diagnosis remain unchanged. The vaccination status is collected in connection with the TBE disease only. There are very few cases with a positive history of TBE vaccination (less than 10); these are usually old individuals with incomplete doses and/or intervals between them.

The antibody prevalence trends in different age groups in both periods suggest that clinically manifest or asymptomatic TBE cases do not induce lifelong immunity, but they are likely to reflect the previous epidemiological situation (in the previous decade?). This hypothesis is also confirmed by the following study. In an area with known high long-term prevalence of TBE in the South Bohemian Region, Czech Republic, 280 permanent residents were screened for anti-TBEV antibodies. Among others, TBE medical history data were collected. Anti-TBEV antibodies were detected in 15.7% of persons with laboratory-confirmed TBE history, known not to be vaccinated against TBE. Another 15.3% developed the antibodies after previous vaccination (Lunackova et al. 2003).

TBE upsurge in the Czech Republic reported in the 1990s affected all age groups (Daniel et al. 2011, Kříž 2012) and thus must have been reflected in the anti–TBEV antibody status of the population. The average positivity rate of 22.4% in the adolescent and adult age groups in year 2001 is in accordance with the data reported by other countries. Below, we present some examples of results from other European countries. It should be realized that TBE, initially known as Central European tick-borne encephalitis, is the infection mainly affecting the countries around the Czech Republic. Baltic states have a different epidemiological situation because of a different tick vector. The ecosystems in some western or southern countries are different as well. Gustafson (1994) reported that out of 903 nonimmunized individuals living in different Stockholm areas TBE seropositivity was detected in 4–22%. In sera from 1261 forestry workers and 233 farmers from the Lublin Region in eastern Poland, anti-TBEV antibodies were detected in 19.8% and 32.0%, respectively (Cisak et al. 1998).

In another study from the Lublin province, the TBE antibody status of the agricultural population was investigated using sera from 242 farmers. Fifty healthy blood donors were used as controls. A significantly higher rate of positives, i.e., 17.3%, was found in the group of farmers than in controls (6.0%) (p<0.05) (Cisak et al. 2003).

In a study conducted in the Warmia-Masuria province of Poland, 878 patients with neurological disorders were screened during 2006–2010. Specific immunoglobulin M (IgM) and IgG anti-TBEV antibodies were detected in 15.5% of these patients, indicating their contact with TBEV (Kubiak et al. 2011).

On the other hand, sera from 412 forestry, agricultural, and outdoor workers screened for anti-TBEV antibodies in Tuscany, Italy, showed low prevalence. Only one worker was positive and other five had borderline values (Tomao et al. 2005). Recently, 461 sera collected from blood donors living in the county of Østfold, Norway, were tested for TBE antibodies. Seroprevalence was 0.65% (Larsen et al. 2014). Seroprevalence of TBE in workers at risk in Alsace, France, was 5.5% (Thorin et al. 2008).

The incidence rate of TBE is known to be higher in males than females (Daniel et al. 2011). This fact is not supported by the antibody levels, which are practically the same in both periods. There is no difference in the treatment of both sexes. In the children's age groups, there is no difference between the incidences of both sexes. The difference starts approximately after 10 years of age and the difference between genders is gradually extending; maximum difference is in the oldest male groups above 55, which moreover are numerous (Kriz et al. 2013). Theoretically, older males may react less immunologically to the antigenic impulse of the TBE infection. It is known that old individuals respond to any vaccination at a lower level as compared to the young ones. Explanation of this finding should be a matter of the further studies.

Conclusions

Sera of healthy individuals aged 10–59 years collected in the 1980s and in 2001 and stored in freezers at −20°C were screened for anti-TBEV antibodies by the virus neutralization test. The seroprevalence rates were 11.5% in the 1980s and 26.3% in 2001. From the logistic regression model, it follows that the chance of detecting anti-TBEV antibodies was more than two times higher in 2001 than in 1978–1989 (OR=2.22). The differences between males and females were not statistically significant nor was sex–period interaction. The time trends in the seropositivity rates were similar all age groups, with the exception of the 10- to 14-year-olds. The rate of seropositives in the age group 15–59 years increased 1.9 times, whereas that in the age group 10–14 years rose 5.1 times.

Footnotes

Acknowledgement

This study was partly supported by the Czech Ministry of Health project grant no. 11425-5/2010.

Author Disclosure Statement

No competing financial interests exist.

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