Objective Geographical differences of Borrelia genospecies and difficulties with comparison of serological findings reported in various regions of the world，led the authors to the implementation of a pilot study on the domiciliary population.The goal of the study was to verify the levels of anti-borrelia antibodies in the population of healthy blood donors and inhabitants living and working in the region with high incidence of ticks.Methods In the sample of Prague blood donors （control sample），total of 200 probands were examined，thereof 100 males at mean age of 33.5 years （range 18～58 years） and 100 females in mean age of 24.4 years （range 18～60 years）.Total of 71 probands were examined in the risk group （forest workers，foresters and tractor drivers working in the forest） from umava forest district Trhanov （West Bohemia），39 males of whom were at mean age of 40.6 years （range 22～57 years） and 32 females at mean age of 44.5 years （range 27～61 years）.Results In all samples，method ELISA NRLB KC 90 was used.Normal values for this method are up to 900 for IgM and up to 700 in IgG.Western blot was used for the confirmation.The criterion for positive WB in the IgM class was the presence of at least 2 positively colored stripes of nitrocellulose membrane and presence at least four stripes in the IgG class.Conclusions Statistically significant difference was found between 2 samples in total distribution of IgM class antibodies determined by means of ELISA method （without relation to the normal values）.Higher level of antibodies in IgM class using ELISA method were detected in the risk group of the forest workers.However，statistically significant difference in the frequence of variable defined as non-normal value was found in IgM class using Western blot method.Statistically significant higher frequency of the tick bites was found in the probands of the risk sample - group of the forest workers.
【关键词】  Lyme borreliosis anti-borrelia antibodies blood donors risk population

　　INTRODUCTION

    Despite many new information about Lyme disease （LD），many questions remain unanswered.Moreover，it is not clear，whether it is possible to transfer experience from other states into home circumstances or to do so only with a certain modification.This specificity （opposed to civilization diseases） is due to the character of the disease （infectious process），but also due to complexicity of its transfer （anthropozoonosis）.In Europe and apparently in Bohemia，different sorts of ticks occur，and mainly different stems of Borrelias.This ensues，above all， the necessity of careful verification of the foreign experience in our conditions.

    Therefore we decided to support some literary information by our observation and designed our ownstudy aimed at determination of anti-borrelia antibodies （AB） in population:a） health blood donors,b） inhabitants living and working in the region of high occurrence of the ticks.

    Prevalence of Anti-borrelia Antibodies in Population

    Geographic differences，endemic regions

    In the last years，many studies appeared in the literature，aiming at comparison of occurrence of AB in the healthy population and in the inhabitant of endemic region，i.e.in risk population，in which in addition to the forest and agricultural workers and foresters，orientation runners are included as well.While in USA，positivity of AB ranges from 0 to 10% ［1-5］ and in England from 1% to 7% ［6］，in Germany was found in 15.7% of rural inhabitants and from 13.7% to 27% in forest workers ［7-9］.Blaauw,et al.examined 431 Dutch foresters;336 out of them （78%） had no complaints in case history compatible with LD，but had positive serological tests，and 65 out of them （15.1%） had no clinical manifestation of LD，but had positive serological findings，only 1.9% from this sample had undoubted or probable symptoms of LD ［10］.Similar findings were reported by other authors ［11］.Berglund,et al.prospectively followed （from 1991 till 1994） 301 residents living in a highly endemic region of Sweden.IgG antibodies were proved by means of ELISA at the beginning of the study in 63 out of them （21%）.In the same sample of probands，increase of AB occurred three years later in 101 out of them （34%）.Apparent LD developed during the follow-up period in total 34 （11%） followed subjects ［12］.

    Risk Population Groups

    In addition to the groups at risk due to occupation，long-term stay in epidemiologically exposed regions，and in addition to the orientation runners，who are exposed to Bb exposed intermittently，a portion of urban population is jeopardized，in a way.Rees′s study is the argument for this;with the aim to specify the data about risk of LD prevalence in urban population，the author inquired of 44 workers of two London parks and 27 caregivers in the zoo outside the city （control sample） about tick bite，symptoms of LD and determined levels of anti-borrelia antibodies.ELISA method proved that the levels in urban park workers  were always significantly higher than in those from control sample ［13］.

    Limits of Serological Screening

    In 1986 German authors gave proof that increased blood levels of antibodies are one of the indicators of frequency of contact with infection in the population ［14］.The study of Fahrer,et al.from 1991 ［15］ undoubtedly belong to valuable studies aiming at prevention and incidence of clinically apparent and asymptomatic Lyme disease.The authors were interested mainly in estimating of benefit of serological screening as the diagnostic procedure，being aware of the limits of sensitivity and specificity in terms of predictive value of this test.They enrolled onto the study the orientation runners from all regions of Switzerland，and it was found that the region where the races take place，is characteristic by colonization with ticks Ixodes ricinus from 5% to 34%.In the discussion，the authors of the study point out the discrepancy between the findings of American authors and their own findings.While the estimation of ratio between apparent and inapparent LD was 1 ：1 in Great Island study ［16］，the Swiss authors proved that the largest portion of LD cases is clinically silent ［17］.The finding of IgG antibodies in 248 out of 950 orientation country runners （26.1%） was in distinct contrast to control sample comprising 101 subjects， in whom positivity of antibodies occurred  in  3.9%～6.0%.Interestingly，only in 6 persons out of  755 （0.8%），apparent clinical picture of LD developed between spring  and autumn，and in 45 out of these persons who were primarily seronegative，asymptomatic seroconversion occurred.Of course，this finding evokes question what are the consequences of high prevalence of positive finding of anti-borrelia antibodies in relation to then egligible occurrence of clinical forms of LD.

    Aim of Study

    The aim of study was ：1）detect and statisticaly evaluate ：a） difference in distribution of AB levels in the sample of probands from risk and non-risk environment （umava forest workers vs.Prague blood donors） by means of ELISA method,b） Frequency of tick bites in the risk and non-risk samples; 2）determine and statistically evaluate the frequency of probands with increased levels of anti-borrelia antibodies in domiciliary population by means of ELISA method and frequency of probands with non-normal level by means of Western blot （WB） method:a） probands without professional exposure to ticks （Prague blood donors）,b） probands with frequent professional exposure to ticks （forest workers from umava forest district）;3） to assess retrospectively epidemiological and clinical case history focusing LD in both sample of probands.

    MATERIALS AND METHODS

    In the sample of Prague blood donors （control group），total of 200 probands were examined，thereof 100 males at mean age of 33.5 years （range 18～58 years） and 100 females at mean age of 24.4 years （range 18～60 years）.

    In the risk group，we examined 71 probands （forest workers，foresters，and tractor drivers working in the forest） from forest district Trhanov （West Bohemia），including 39 males at mean age of 40.6 years （range 22～57 years） and 32 females at mean age of 44.5 years （range 27～61 years）.

    Blood samples were examined at National reference laboratory for Lyme borreliosis CEM in the State Health Institute in Prague.In all samples，ELISA method NRLB KC90 was used.Normal range for this method is up to 900 for IgM and up to 700 for IgG.Western blot was used for confirmation.Criterion for positive WB at IgM class is the presence of at least two positively colored specific stripes of nitrocellulose membrane and the presence of at least four stripes at IgG class ［18,19］.

    The distribution of values of levels of anti-borrelia antibodies is not symmetrical （gaussian） distribution and therefore we used non-parametric method for the testing-Mann-Whitney test.This test was used for hypothesis testing on equality of distribution of values of antibodies IgM and IgG class by ELISA method irrespective of the fact whether the values were normal or increased.

    χ2 test was used for statistical evaluation of occurrence of variable defined as increased level of antibodies separatedly in classes IgM and IgG determined by ELISA method and occurence of variable defined as non-normal value for Western blot again in classes IgM and IgG.

    RESULTS

    In control sample （200 healthy blood donors from Prague），higher values of anti-borrelia antibodies were proved in 23 （11.5%） out of them.In 6 cases （3%），IgM class anti-borrelia antibodies were increased，in 18 cases （6%） increase in IgG class and 1 proband （0,5%） had higher level of antibodies in either classes.Western blot Lyme method proved higher levels in IgG class in 10 probands （10%）.In IgM class，the results were negative in all cases.

    Positive case history of tick bite during last 6 weeks was in 11 probands （5.5%） from control sample，5 males and 6 females.Only in two out of them skin eruption as possible erythema migrans （one male and one female） appeared，athralgias occurred in three females and chronic fatigue syndrome in one male （Table 1）.

    In the risk group （71 forest workers from umava），higher level of AB were proved in 13 （18.3%） out of them.In 4 cases （5.6%），AB levels were increased in IgM class，in 6 cases （8.5%） in IgG class.One proband （0.7%） had increased levels of antibodies in either classes.WB method proved increased levels in IgM class in 9 probands （12.7%）.Increased levels in IgG class were proved in 5 probands （7%） positive in 1 （1.4%）.

    Ticks in case history during last 6 weeks was found in 10 probands （14.1%） from control sample，5 males and 5 females.Skin eruption as possible erythema migrans occurred only in two females，arthralgia in two females and chronic fatigue syndrome in one female （Table 2）.

    Table 1  Probands from Control Sample （Prague blood donors） in Whom Increased Levels of Anti-borrelia Antibodies Were Proved with Concomitant Occurrence of a Symptom Compatible with Possible Clinical Manifestation of Lyme Disease

    Table 2  Probands from Risk Group （forest workers from umava） in Whom Increased Levels of Anti-borrelia Antibodies Were Proved，with Concomitant Occurence of Symptom Compatible with Possible Clinical Manifestation of Lyme Disease

    Notes:EM=erythema migrans，CSF=chronic syndrome fatigue，

    ELISA=enzyme-linked immunosorbent assay，WB=Western blot，

    +=yes,0=no,+/-=borderline

    Comparison of distribution of levels of anti-borrelia antibodies in both samples is recapitulated in Table 3，4 and in Figure 1，2. Table 3  Basic Statistical Indicators of Values of Antibodies IgM Class

    Table 4  Basic Statistical Indicators of Values of Antibodies IgG Class

    Figure 1  Comparison of distribution of IgM class antibodies

    Figure 2  Comparison of distribution of IgG class antibodies

    Comparison of occurrence of evaluated variables （increased levels of antibodies and occurrence of ticks） in either samples is recapitulated in Table 5.Table 5  Comparison of Occurrence of Evaluated Variables in Both SamplesNote:ELISA=enzyme-linked immunosorbent assay，WB=Western blot

    CONCLUSION

    The comparison proves statistically significant difference in total distribution of IgM class antibodies determined by ELISA method （without relation to normal values）.Higher values of antibodies of IgM class by ELISA method were found in the risk group of forest workers.In IgG class by ELISA method，no significant difference in total distribution of AB between two groups was found，thus the risk group fails to differ from the control group in levels of IgG antibodies by means of ELISA method （without relation to normal values）.

    On determination of difference of frequency of a variable defined as increased level of AB antibodies compared to standard by means of ELISA method，no statistically significant difference between two groups was found，neither in IgM nor in IgG class.Therefore risk group does not differ from control group in terms of number of individuals in comparison with standard with increased levels of IgM class and IgG class detected by ELISA method.

    However，by means of Western blot method，statistically significant difference in frequency of a variable defined as non-normal value in IgM class was found.In IgG class，statistically significant difference in frequency of non-normal values was not found by means of Western blot.

    Statistically significantly higher frequency of tick bites was found in the probands of the risk sample-group of forest workers.

    Following the literature from Europe and USA it is possible to generalize，despite some our experience that:（1）regional differences are not significant，provided there are not high-risk territories or “professional exposure”.（2）There is no significant difference between males and females.（3）Considerable caution is needed in comparing   control and risk groups;condition for such comparison is exact defining of characteristics of both groups.

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（Editor Jaque）