Epidemiology and diagnosis of Lyme borreliosis

The multisystem disease Lyme borreliosis is the most frequent tick‐transmitted disease in the northern hemisphere. In Europe Lyme borreliosis is most frequent in Central Europe and Scandinavia (up to 155 cases per 100,000 individuals) and is caused by the species, B. burgdorferi sensu stricto, B. afzelii and B. garinii. The recently detected genospecies A14S may also play a role in skin manifestations. Microbiological diagnosis in European patients must consider the heterogeneity of borreliae for development of diagnostic tools. According to guidelines of the USA and Germany, serological diagnosis should follow the principle of a two‐step procedure (enzyme‐linked immunosorbent assay (ELISA) as first step, if reactive; followed by immunoblot). The sensitivity and standardization of immunoblots has been considerably enhanced by use of recombinant antigens (p100, p58, p41i, VlsE, OspC, DbpA) including those expressed primarily in vivo (VlsE and DbpA) instead of whole cell lysates. VlsE is the most sensitive antigen for IgG antibody detection, OspC for IgM antibody detection. At present, detection rates for serum antibodies are 20%–50% in stage I, 70%–90% in stage II, and nearly 100% in stage III Lyme disease. Detection of the etiological agent by culture or polymerase chain reaction (PCR) should be confined to specific indications and specialized laboratories. Recommended specimens are skin biopsy specimens, cerebrospinal fluid (CSF) and synovial fluid. The best results are obtained from skin biopsies with culture or PCR (50%–70%) and synovial tissue or fluid (50%–70% with PCR). CSF yields positive results in only 10%–30% of patients except when the duration of symptoms is shorter than 2 weeks (50% sensitivity). Methods which are not recommended or adequately documented for diagnosis are antigen tests on body fluids, PCR of urine, and lymphocyte transformation tests.     

Genospecies and their influence on immunoblot results

In Europe at least three human pathogenic species of Borrelia burgdorferi sensu lato are the causative agents of Lyme borreliosis. All three species have been isolated or detected by PCR from skin, CSF and synovial fluid of patients with skin lesions, neuroborreliosis and Lyme arthritis respectively. Studies using strains representing the three species as antigen for the immunoblot revealed that interpretation criteria depend strictly on the strain used as antigen. More than using certain species as antigen it is important to use strains (f.e. B. afzelii strain PKo) expressing certain immunodominant antigens like OspC and p17 which may not be expressed by other strains in vitro. Using strain PKo as antigen the two band criterium can be used without loss of too much sensitivity compared to using B. burgdorferi sensu stricto strain PKa2 and B. garinii strain PBi. The use of recombinant antigens allows selection of highly specific and combination of homologous antigens from different strains; however not all desirable antigens have been recombinantly expressed. Addition of p17 and p58 as antigens may improve the sensitivity of the hitherto described recombinant antigen immunoblots containing the antigens p83/100, p39, OspC and the p41 internal fragment.     

Molecular diagnosis of Lyme disease: review and meta-analysis.

The diagnosis of Lyme disease is difficult because tests that reflect active disease or have reasonable sensitivity and specificity are lacking or not timely. Molecular methods are controversial because of differences in assays, gene targets, and limited clinical validation. This review summarizes published assays for Lyme disease diagnosis using skin, plasma, synovial fluid, cerebrospinal fluid (CSF), and urine. Meta-analyses show the strengths and weaknesses of these methods. Overall, assays for skin and synovial fluid (68% and 73%, respectively) have high sensitivity and uniformity. The low test sensitivity of CSF (18%) and plasma (29%), variable sensitivities among CSF and urine assays, and persistence of Borrelia burgdorferi DNA in urine and synovial fluid even with therapy and convalescence make these unsuitable for primary diagnosis. Molecular assays for Lyme disease are best used with other diagnostic methods and only in situations in which the clinical probability of Lyme disease is high.     

Improvement in the laboratory recognition of lyme borreliosis with the combination of culture and PCR methods.

BACKGROUND: Lyme disease is a multisystem, multistage infection caused by three genospecies of the Borrelia burgdorferi sensu lato species. The diagnosis of Lyme disease is based on a history of tick-bite, physical examination, and serological tests. In the seronegative patients with Lyme borreliosis symptoms, additional testing should be introduced. METHODS: The study group was composed of 240 hospitalized patients presented with various clinical symptoms suggesting Lyme borreliosis: 221 of the patients with neurological abnormalities and 19 with oligoarticular arthritis. Citrated blood and serum samples were collected from the patients for culture and serological examination, respectively. Moreover, 173 cerebrospinal and 6 synovial fluid samples were tested. New oligonucleotide primers based on B. burgdorferi sensu lato 16SrRNA gene sequences were designed for the detection of the bacteria in blood, cerebrospinal, and synovial fluid specimens with PCR. Levels of specific antibodies were measured in serum, cerebrospinal fluid and synovial fluid samples using ELISA and Western blot. B. burgdorferi spirochetes from blood, cerebrospinal fluid, and synovial fluid samples were cultured in cell line. Extracted and purified B. burgdorferi DNA was identified by PCR with new oligonucleotide primers. Then three genospecies were identified by PCR amplification with other primer sets specific for 16S rDNA and/or by the restriction fragment length polymorphism of 23S(rrl)-5S(rrf). RESULTS: Bacterial DNA were found in samples from 32 patients, including 28 patients with neuroborreliosis and 4 with Lyme arthritis. B. burgdorferi-specific IgM and/or IgG serum antibodies were detected in 14 of these patients. Fourteen strains of Borrelia garini, 4 strains of Borrelia afzelii and 1 strain of B. burgdorferi sensu stricto were identified by PCR. Genospecies were not recognized in 13 specimens. CONCLUSIONS: The procedure can be a rapid and sensitive diagnostic method for the detection of etiological agents in clinical materials derived from patients with the clinical symptoms of Lyme borreliosis. It can be utilized for both basic research as well as routine laboratory diagnosis.     

Limitation of serological testing for Lyme borreliosis: evaluation of ELISA and western blot in comparison with PCR and culture methods

The aim of the study was to evaluate a one-step procedure using an ELISA test of high specificity and a two-step procedure using immunoblot as a confirmation test, and to compare the results of serological testing with detection of bacterial DNA and living spirochetes. Sera, synovial (SF) and cerebro-spinal fluids (CSF) were obtained from 90 patients with clinical symptoms of Lyme borreliosis. Serum samples were tested with recombinant ELISA and Western blot assay. Citrated blood, cerebrospinal and synovial fluids samples were cultured in cell line and tested by PCR to detect spirochetes. No correlation was found between levels of specific B. burgdorferi antibodies detected with a recombinant antigen ELISA and the number of protein fractions developed with these antibodies by immunoblot. Moreover, Lyme borreliosis patients who have live spirochetes in body fluids have low or negative levels of borrelial antibodies in their sera. This indicates that an efficient diagnosis of Lyme borreliosis has to be based on a combination of various techniques such as serology, PCR and culture, not solely on serology.     

Simultaneous expression of Borrelia OspA and OspC and IgM response in cerebrospinal fluid in early neurologic Lyme disease.

Lyme disease is the major tick-borne disease, caused by Borrelia burgdorferi (Bb). Neurological involvement is common in all stages. In vivo expression of Bb antigens (Ags) and the immune response to them has not been well investigated in the cerebrospinal fluid (CSF). Upregulation of outer surface protein (Osp) C and concomitant downregulation of OspA before tick inoculation of the spirochete has been reported in skin and blood in animals. CSF OspA Ag in early disease suggests otherwise in CSF. Early Ag expression and IgM response in human CSF was investigated here. Paired CSF and serum was collected from 16 early, predominantly erythema migrans Lyme disease patients with neurologic problems, 13 late Lyme disease patients, and 19 other neurologic disease (OND) controls. Samples were examined for IgM reactivity to recombinant Bb-specific Osps using ELISA and immunoblot. Of 12 early Lyme disease patients with neurologic involvement with both CSF and serum IgM against OspC, 7 (58%) had IgM to OspA (n = 5) or OspB (n = 2) that was restricted to the CSF, not serum. Overall, 12 of 16 (75%) of these early Lyme disease patients with neurologic involvement had CSF and serum IgM against OspC. Only 3 of 13 (23%) late Lyme disease patients and none of 19 OND controls had CSF IgM directed against OspC. In conclusion, in CSF, OspC and OspA can be coexpressed, and IgM response to them occurs in early Lyme disease patients with neurologic involvement. This biologic finding may also provide a discriminating marker for CNS infection in Lyme disease.     

Procalcitonin levels in patients with Lyme borreliosis

BACKGROUND: Serum and cerebrospinal fluid (CSF) procalcitonin levels were assessed and compared for different groups of patients with Lyme borreliosis. PATIENTS AND METHODS: 50 adult patients with Lyme borreliosis, referred to our department from March to June 2001, were included in this prospective study. Patients were divided into three groups. The first group consisted of 20 consecutive patients with typical solitary erythema migrans, representing early localised Lyme borreliosis, the second group comprised 20 patients with early disseminated Lyme borreliosis (10 with multiple erythema migrans and 10 with neuroborreliosis), and 10 patients with acrodermatitis chronica athrophicans represented the group with chronic Lyme borreliosis. Blood specimens were taken from all patients included in the study, but CSF samples were restricted to those with disseminated and chronic Lyme borreliosis. The serum and CSF procalcitonin levels were determined utilizing the LUMI PCT (an immunoluminometric assay using two antigen-specific monoclonal antibodies). RESULTS: Serum and CSF procalcitonin levels were in normal range in the large majority of patients. The levels of serum procalcitonin did not differ in the three groups of patients with Lyme borreliosis (p = 0.5006). The corresponding values for patients with solitary erythema migrans (early localised Lyme borreliosis), early disseminated Lyme borreliosis, and chronic Lyme borreliosis were 0.26 (0.11-0.43), 0.22 (0.10-0.67), and 0.28 (0.13-0.66) microgram/ml, respectively. Moreover, procalcitonin levels in CSF were also low and comparable for patients with multiple erythema migrans (median 0.38, range 0.24-0.54 microgram/ml), neuroborreliosis (median 0.16, range 0.10-0.47 microgram/ml), and acrodermatitis chronica athrophicans (median 0.30, range 0.15-0.45 microgram/ml). The differences were not statistically significant (p = 0.7579). CONCLUSIONS: In the large majority of patients with Lyme borreliosis procalcitonin values are within normal range. Serum and CSF procalcitonin levels are of no value for differentiation between early localised, early disseminated and chronic Lyme borreliosis.     

Detection of Borrelia burgdorferi DNA by polymerase chain reaction in the urine and breast milk of patients with Lyme borreliosis

Current laboratory diagnosis of Lyme borreliosis relies on tests for the detection of antibodies to Borrelia burgdorferi with known limitations. By using a simple extraction procedure for urine samples, B. burgdorferi DNA was amplified by a nested PCR with primers that target the specific part of the flagellin gene. To control possible inhibition of the enzyme (polymerase), a special assay using the same primers was developed. We examined 403 urine samples from 185 patients with skin manifestations of Lyme borreliosis. Before treatment, B. burgdorferi DNA was detected in 88 of 97 patients with Lyme borreliosis. After treatment, all but seven patients became nonreactive. Six of these seven persons suffered from intermittent migratory arthralgias or myalgias, and one from acrodermatitis chronica atrophicans. Two of 49 control patients with various dermatologic disorders and none out of 22 presumably healthy persons were reactive in the PCR. In addition to urine, breast milk from two lactating women with erythema migrans was tested and also found reactive. Borrelia burgdorferi DNA can be detected with high sensitivity (91%) by a nested PCR in urine of patients with Lyme borreliosis. In addition, this test can be a reliable marker for the efficacy of treatment.     

Serodiagnosis of Lyme borreliosis infection using surface plasmon resonance

BACKGROUND: Label-free biosensors are ideally suited for the direct monitoring of binding events without the need for additional labeling substances; however, their application in the field of serodiagnosis is not trivial. The major problem is the unspecific adsorption of blood serum components to the sensor surface. METHODS: A surface plasmon resonance (SPR) sensor has been used for the direct detection of Lyme borreliosis specific antibodies in blood serum. The combination of an optimal dilution factor with the addition of suitable detergents minimizes the unspecific adsorption. Serum samples from healthy donors and infected patients have been analyzed and the results were compared with a certified immunoassay and a western blot. RESULTS: A serum dilution of 1:20 in HBS-buffer with 0.05% Tween20 and 1 mg/mL carboxymethyl dextran reduces unspecific adsorption significantly and enables the identification of antibodies against the OspC/pepC10 antigen pair with a sensitivity of 92% and that against the VlsE/C6 pair with 81% sensitivity; the specificities are 82% and 86% respectively. Positive hits in the western blot could also be determined in the SPR-assay with a correlation of 96.5%. CONCLUSION: The presented optical label-free technique has the potential for a precise and fast identification of pathogen-specific antibodies without the need for a secondary labeling antibody.     

Serologic evidence for tick-borne pathogens other than Borrelia burgdorferi (TOBB) in Lyme borreliosis patients from midwestern Germany

The seroprevalence of antibodies against the human granulocytic ehrlichiosis agent (HGE) and Babesia microti was retrospectively determined in 76 Lyme borreliosis patients and in 44 asymptomatic individuals with a positive borreliosis serology, in comparison to 100 healthy blood donors from the Rhein-Main area. Additionally, seroreactivity for tick-borne encephalitis virus (TBEV) was investigated. For antibody detection, commercially available immunofluorescence assays (MRL Diagnostics, USA) and a TBEV-ELISA (Immuno, Germany) were used. In the control group, the positivity rate for anti-Borrelia burgdorferi (IgG/IgM) and anti-Babesia microti-antibodies in the population of the Rhein-Main area (Midwestern Germany) may be estimated at 15% and 8%, respectively. Examination for both HGE and TBEV demonstrated seroreactivity (IgG) in 1% of tested individuals. Specific anti-HGE IgG and/or IgM antibodies were more often discovered in cases of early Borrelia infection (stage I: 13.6%, stage II: 18.4%) than in patients with stage III disease (0%) or in seropositive but asymptomatic patients (6.8%). Investigation for TBEV revealed seroreactivity for IgG in 13% of these cases. No TBEV-IgM was found. Interestingly, the prevalence of anti-HGE and anti-TBEV antibodies among Lyme borreliosis patients and seropositive patients without active Lyme disease symptoms was significantly higher than that in the control group of healthy blood donors (p < 0.05). Likewise, antibody titers reflecting a recent infection with Babesia microti could be demonstrated more often in patients with Lyme borreliosis stage I or II (p < 0.05). Analysis of 50 samples from patients with florid or recent syphilis infection revealed no crossreactivity between Babesia microti, HGE and Treponema pallidum. Our findings suggest that concomitant or serial infection due to TOBB may be common in tick exposed patients from the Rhein-Main area and in European countries in general. Hence, in addition to TBEV, human babesiosis and HGE should always be considered by European physicians in the differential diagnosis of acute febrile illness following a tick bite.     

Serodiagnosis of Lyme borreliosis using detection of different immunoglobulin (sub)classes by enzyme-linked immunosorbent assay and Western blotting

To improve the performance of enzyme-linked immunosorbent assays for the serodiagnosis of Lyme borreliosis, the prevalence of several immunoglobulin classes and subclasses against various antigens of Borrelia burgdorferi was investigated by Western blotting. The sera of 40 early Lyme borreliosis patients (ELB), 27 late Lyme borreliosis patients (LLB), 62 healthy controls and 140 non-Lyme borreliosis patients were used. Detection of IgG1 versus total IgG was found to be more sensitive in detecting Borrelia burgdorferi antigens, especially flagellin (41 kD) protein, but did not improve the performance of Western blotting. The use of IgG1 detection showed an increase in sensitivity and specificity for the early Lyme borreliosis patient group compared to the standard IgG and IgM detection method by enzyme immunoassays using purified Borrelia burgdorferi flagellum. However, in an enzyme immunoassay using a total sonicate, sensitivity in detecting early Lyme borreliosis and late Lyme borreliosis with IgG1 remained lower compared to the detection of early Lyme borreliosis by IgM antibodies and late Lyme borreliosis by total IgG antibodies.     

Principles of the diagnosis and antibiotic treatment of Lyme borreliosis

Clinical signs and symptoms are an essential aspect of the diagnosis of Lyme borreliosis. Thus, a thorough knowledge of the clinical features of the disease is important. Established clinical definitions could be of help in everyday clinical practice and especially to compare the findings of different authors or groups. The characteristic sign that permits the diagnosis of Lyme borreliosis is a typical erythema migrans skin lesion. Highly suggestive manifestations are ear lobe lymphocytoma, acrodermatitis chronica atrophicans and Bannwarth's syndrome. The majority of other signs and symptoms are only suggestive and, when expressed individually, may have a very limited or even symbolic value for the purpose of diagnosis. Laboratory confirmation of borrelial infection is needed, as a rule, for all manifestations of Lyme borreliosis with the exception of typical erythema migrans. In clinical practice indirect laboratory methods are usually employed. Determination of borrelial IgM and IgG antibodies by immunofluorescence assays or enzyme-linked immunosorbent assays has not been standardised, and correlation of the results from different laboratories and/or different commercial tests may be poor. Immunoblotting may solve some of the many dilemmas but could (especially in Europe) raise additional questions in a field in which numerous uncertainties already exist. The reliability of methods for direct detection of borrelial infection other than culture to ascertain spirochetes in tissue specimens is open to question. Treatment with antibiotics is reasonable in all stages of Lyme borreliosis and for all clinical manifestations; however, it has been most effective early in the course of the illness. The choice of antibiotic depends upon many factors including the efficacy, pharmacokinetic profile, side effects, expected compliance and price. For the majority of manifestations the most effective antibiotic, the optimal dosage, and the most appropriate duration of treatment have not been exactly determined. Recommendations for the treatment of Lyme borreliosis in Slovenia are presented.     

Antibodies to Borrelia burgdorferi in European populations

We compared the antibodies to B. burgdorferi in three different populations in order to evaluate the diagnostic reliability of Lyme borreliosis serologic analysis. The subjects included 25 patients with Lyme borreliosis (Group 1); 50 patients with diseases of unknown cause, B. burgdorferi ELISA-positive in serum and without B. burgdorferi infection (Group 2); and 1,251 individuals without Lyme borreliosis (Group 3). All samples were tested for B. burgdorferi B31 and B. afzelii antigens using ELISA. The positive results of the ELISA B. burgdorferi B31 assay were confirmed with Western blot for the same strain. In Group 3, 162 (12.9%) patients were ELISA positive for B. burgdorferi B31, while only 6 (0.6%) patients had IgG ELISA antibodies to B afzelii. Bands in WB were detected in 104 (8.4%) of the Group 3 subjects. The bands found to be most reliable for the identification of strain B. burgdorferi B31 by IgG WB were those representing the 93, 39, 34, and 23-kDa proteins. Our results show that serologic diagnosis of Lyme borreliosis is far from clearly established. To date, the only reliable criteria are clinical ones correlated with laboratory evidence.     

Recent advances in the diagnosis of Lyme disease

Diagnosis of human Lyme borreliosis is usually based on serology, which has a number of pitfalls. In the early phase of the disease serology can still be negative, whereas false-positive results are also common. The interpretation of confirmatory Western blot tests is not always easy. Furthermore, routine serology cannot discriminate between active and past infection. In addition, recombinant antigens are being introduced to improve serologic tests. New developments in the diagnosis of Lyme disease are the development of PCR tests. This review gives an overview of the molecular diagnostic possibilities of Lyme borreliosis, mainly by PCR, and describes some interesting possibilities for future serology.     

Evaluation of in vivo expressed Borrelia burgdorferi antigens for improved IgM serodiagnosis of early Lyme disease

Improved serologic tests are needed for accurate diagnosis and proper treatment of early stage Lyme disease. We evaluated the 3 antigens currently used for 2-tiered IgM immunoblot testing (FlaB, OspC, and BmpA) in combination with 3 additional antigens (BBA65, BBA70, and BBA73) and measured the sensitivity and specificity against a serum repository of positive and negative controls. Using 3 statistical methods for positivity cutoff determinations and scoring criteria, we found increased sensitivities for early Lyme disease when 2 of 6 antigens were positive as compared with the 2 of 3 antigen IgM criteria currently used for second-tier immunoblot scoring. Specificities for negative controls were comparable or superior to using 2 of 3 antigens. These results indicate that IgM sensitivity and specificity of serological testing for Lyme disease in the early stages of illness can be improved by employing antigens that target the initial host antibody responses.     

Lyme disease—another transfusion risk?

Lyme disease (or Lyme borreliosis) is caused by a spirochetal bacteria, Borrelia burgdorferi. Increased recognition of the disease and increased exposure to the vector (ticks) capable of spreading B. burgdorferi from animal hosts have resulted in a rise in the number of cases of Lyme borreliosis reported in the United States. There are three stages of the clinical course of Lyme borreliosis; however, not all those infected will have typical manifestations of each stage, such as the arthritis of the third stage. Routine blood cultures will rarely document bacteremia and serologic testing is not yet reliable. Early treatment can prevent later stages of Lyme borreliosis. There is evidence that transmission of B. burgdorferi by blood transfusion is possible, but, to date, there has been no documentation of transfusion- associated Lyme borreliosis. Thus, no new recommendations for screening donors to identify possible carriers of B. burgdorferi are suggested at this time.     

Late stage Lyme borreliosis in children

These cases illustrate that late stage Lyme borreliosis can occur in children without a history of tick bite or ECM; this disorder can manifest itself initially as a seventh cranial nerve palsy, heart block, or arthritis, and the arthritis syndrome can mimic oligoarticular juvenile rheumatoid arthritis. The diagnosis of Lyme borreliosis depends upon clinical recognition. In the absence of ECM, tests for antibodies to Borrelia burgdorferi can provide an invaluable tool in assisting in the diagnosis. Children who live in or visit areas endemic for Lyme borreliosis and who have arthritis, heart block, or neurologic disorders such as facial palsy should be tested for antibodies to Borrelia burgdorferi if no other cause for the disease syndrome is identified clinically.     

Lyme disease--another transfusion risk?

Lyme disease (or Lyme borreliosis) is caused by a spirochetal bacteria, Borrelia burgdorferi. Increased recognition of the disease and increased exposure to the vector (ticks) capable of spreading B. burgdorferi from animal hosts have resulted in a rise in the number of cases of Lyme borreliosis reported in the United States. There are three stages of the clinical course of Lyme borreliosis; however, not all those infected will have typical manifestations of each stage, such as the arthritis of the third stage. Routine blood cultures will rarely document bacteremia and serologic testing is not yet reliable. Early treatment can prevent later stages of Lyme borreliosis. There is evidence that transmission of B. burgdorferi by blood transfusion is possible, but, to date, there has been no documentation of transfusion-associated Lyme borreliosis. Thus, no new recommendations for screening donors to identify possible carriers of B. burgdorferi are suggested at this time.     

Utility of a commercial immunoblot kit (BAG-Borrelia blot) in the diagnosis of the preliminary stages of Lyme disease

The aim of this study was to evaluate the usefulness of a commercial immunoblot (IgG and IgM BAG-Borrelia blot) in the serologic diagnosis of the early stages of Lyme disease. A total of 42 sera from patients with Lyme disease (24 patients with localized early stage (LES) and 18 patients with disseminated early stage (DES)) and 129 sera from patients with non-Lyme diseases (specificity control sera) were studied. IgG anti-p41 from Borrelia burgdorferi s.l. was present in 95.2% of patients followed by anti-p41/I PBi (16.7%), anti-p100 (9.5%) and anti-OspA (9.5%). IgM anti-p41 was present in 66.7% of patients, p41/iPBi (54.8%) and OspC (33.3%). IgM against p100, OspA and OspC were more frequent in DES patients (16.7%, 27.8% and 44.4%) than in LES patients (0.0%, 4.2% and 25.0%). In 4.8% of the cases no IgG bands were present and in 26.2% no IgM bands were present. With the exception of isolated p41 bands (59.5%), no band pattern exceeded 17%. Using manufacturer's instructions, test sensitivity in diagnosis of the early stage of Lyme disease is 61.9%, specificity 98.4% and positive and negative predictive values 92.8% and 88.8% respectively. Applying the EUCALB 5, 6 or 7 rules sensitivity increased to 73.8% although specificity decreased to 89.9%. Of the 129 specific control sera, 41.8% presented IgG anti-p41 and 10.8% IgM anti-p41. Patients with non-Lyme diseases that presented more IgG and IgM bands were those patients with syphilis (88.2%), patients with anti-HIV antibodies (57.8%) and patients with anti-nuclear antibodies (ANA) (52.3%).