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2.
Arch Intern Med ; 161(16): 2015-20, 2001 Sep 10.
Article in English | MEDLINE | ID: mdl-11525704

ABSTRACT

BACKGROUND: The present recommendation for the serologic diagnosis of Lyme disease is a 2-tier process in which a serum sample with a positive or equivocal result by an enzyme-linked immunosorbent assay (ELISA) or immunofluorescent assay is then followed by supplemental testing by Western blot. Our laboratory has developed recombinant chimeric proteins composed of key Borrelia epitopes. These novel antigens are consistent and are easily standardized. METHODS: We adapted these recombinant proteins into a new immunochromatographic format that can be used as a highly sensitive and specific first-tier assay that can be used to replace the ELISA or immunofluorescent assay. RESULTS: This rapid test was equally sensitive (P>.05) and more specific (P<.05) than a frequently used commercial whole cell ELISA. The overall clinical accuracy achieved on agreement studies among 3 Lyme research laboratories on clinically defined serum panels was shown to be statistically equivalent to the commercial ELISA. The assay can detect anti-Borrelia burgdorferi antibodies in either serum or whole blood. CONCLUSION: This sensitive and specific rapid assay, which is suited for the physician's office, streamlines the 2-tier system by allowing the physician to determine if a Western blot is necessary at the time of the initial office visit.


Subject(s)
Antibodies, Bacterial/blood , Borrelia Infections/diagnosis , Borrelia burgdorferi Group/immunology , Chromatography , Epitopes/analysis , Recombinant Fusion Proteins/analysis , Blotting, Western , Borrelia Infections/immunology , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Humans , Sensitivity and Specificity
4.
J Clin Microbiol ; 38(7): 2530-5, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10878038

ABSTRACT

Current serologic Lyme disease tests use whole borrelia cells as the source of antigen. These assays are difficult to standardize and to optimize for sensitivity and specificity. To help solve these problems, we constructed a library of recombinant chimeric proteins composed of portions of key antigens of Borrelia burgdorferi. These proteins were then used to develop an enzyme-linked immunosorbent assay. We compared our assay with the most sensitive of three whole-cell borrelia assays. We found that the recombinant assay could detect antibodies significantly better from early Lyme disease sera (P<0.05), and had the same sensitivity for late Lyme disease sera, as the most sensitive whole-cell borrelia assay. On potentially cross-reactive sera, the recombinant assay was more specific, but not significantly so, than the best whole-cell borrelia assay. Optimization of the recombinant assay offers the potential for a significant improvement in both sensitivity and specificity.


Subject(s)
Antibodies, Bacterial/blood , Antigens, Bacterial/immunology , Borrelia burgdorferi Group/immunology , Enzyme-Linked Immunosorbent Assay/methods , Lyme Disease/diagnosis , Recombinant Fusion Proteins/immunology , Antigens, Bacterial/genetics , Antigens, Bacterial/metabolism , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/immunology , Bacterial Outer Membrane Proteins/metabolism , Flagellin/genetics , Flagellin/immunology , Flagellin/metabolism , Humans , Lyme Disease/microbiology , Reagent Kits, Diagnostic , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sensitivity and Specificity
5.
Arch Dermatol ; 135(11): 1329-33, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10566830

ABSTRACT

OBJECTIVE: To assess human skin biopsy specimens from erythema migrans lesions for the presence of infection with multiple strains of the Lyme disease spirochete, Borrelia burgdorferi. DESIGN: Skin biopsy specimens were obtained prospectively from patients with erythema migrans. To determine allelic differences and strain identification of B burgdorferi, the biopsy specimens were analyzed by cold single-strand conformation polymorphism of an amplified fragment of the outer surface protein C (ospC) gene. Further single-strand conformation polymorphism patterns of amplified ospC genes from culture isolates were compared with polymerase chain reaction products obtained directly from erythema migrans biopsy specimens. SETTING: A private dermatology office and a university medical center outpatient department. PATIENTS: Sixteen patients presenting with erythema migrans. RESULTS: Two of the 16 patients in this cohort were infected with 2 B burgdorferi sensu stricto strains, as evidenced by 2 ospC alleles in their skin biopsy results. CONCLUSION: This is the first documented description of the existence of more than a single strain of B burgdorferi sensu stricto in a human specimen.


Subject(s)
Antigens, Bacterial , Borrelia burgdorferi Group/classification , Borrelia burgdorferi , Erythema Chronicum Migrans/microbiology , Lyme Disease/microbiology , Adult , Alleles , Bacterial Outer Membrane Proteins/genetics , Biopsy , Borrelia burgdorferi Group/genetics , Cohort Studies , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Gene Expression Regulation, Bacterial , Humans , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Prospective Studies , Sequence Analysis, DNA , Skin/microbiology
7.
Infect Immun ; 67(7): 3518-24, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10377134

ABSTRACT

Lyme disease begins at the site of a tick bite, producing a primary infection with spread of the organism to secondary sites occurring early in the course of infection. A major outer surface protein expressed by the spirochete early in infection is outer surface protein C (OspC). In Borrelia burgdorferi sensu stricto, OspC is highly variable. Based on sequence divergence, alleles of ospC can be divided into 21 major groups. To assess whether strain differences defined by ospC group are linked to invasiveness and pathogenicity, we compared the frequency distributions of major ospC groups from ticks, from the primary erythema migrans skin lesion, and from secondary sites, principally from blood and spinal fluid. The frequency distribution of ospC groups from ticks is significantly different from that from primary sites, which in turn is significantly different from that from secondary sites. The major groups A, B, I, and K had higher frequencies in the primary sites than in ticks and were the only groups found in secondary sites. We define three categories of major ospC groups: one that is common in ticks but very rarely if ever causes human disease, a second that causes only local infection at the tick bite site, and a third that causes systemic disease. The finding that all systemic B. burgdorferi sensu stricto infections are associated with four ospC groups has importance in the diagnosis, treatment, and prevention of Lyme disease.


Subject(s)
Antigens, Bacterial , Bacterial Outer Membrane Proteins/genetics , Borrelia burgdorferi Group/genetics , Borrelia burgdorferi , Lyme Disease/microbiology , Borrelia burgdorferi Group/pathogenicity , Genes, Bacterial , Genetic Variation , Humans , Virulence/genetics
8.
Acta Med Austriaca ; 25(2): 44-50, 1998.
Article in German | MEDLINE | ID: mdl-9681041

ABSTRACT

Borrelia burgdorferi infection (BBI) is suggested to be associated with dilated cardiomyopathy (IDC). Stanek et al. were able to cultivate Borrelia burgdorferi (BB) from myocardial biopsy tissue of a patient with longstanding dilated cardiomyopathy. Here we present a study in which we examined the effect of standard antibiotic treatment on the left ventricular ejection fraction (LV-EF) in patients with dilated cardiomyopathy associated with BBI. In this study we assessed the serum (IgG, IgM ELISA; Western Blot) and the history of 46 IDC-patients with specific respect spect to BBI (mean LV-EF: 30.4 +/- 1.3%; measured by cardiac catheterisation and echocardiography--length-area-volume method). All 46 patients received standard treatment for dilated cardiomyopathy: ACE-inhibitors, digitalis and diuretics. 11 (24%) patients showed positive serology and a history of BBI; 9 of these also had a typical history of tick bite and erythema chronicum migrans (ECM) and/or other organ involvement, 2 had no recollection of tick bite or EMC, but showed other BB-associated disorders (neuropathy, oligoarthritis). These 11 patients with BBI received standard antibiotic treatment with intravenous ceftriaxone 2 g bid for 14 days. 6 (55%) recovered completely and showed a normal LV-EF after 6 months, 3 (27%) improved their LV-EF and 2 (18%) did not improve at all. This amounts to 9 (82%) recovery/improvement in the BB-group. The 35 patients who did not show positive serology or a history of BBI did not receive antibiotic treatment. In this group without BBI 12 (26%) showed recovery/improvement following the standard treatment of dilated cardiomyopathy (see above). Our results indicate that BBI could play a decisive role in the development of dilated cardiomyopathy, especially in a geographical region as Graz, where BB is endemic. While aware of the small number of BB-patients in this study, we nevertheless conclude that, in a remarkable number of patients with signs of BBI, dilated cardiomyopathy could be reversed and LV-EF improved upon standard antibiotic treatment.


Subject(s)
Cardiomyopathy, Dilated/diagnosis , Lyme Disease/diagnosis , Myocardial Contraction/physiology , Adolescent , Adult , Aged , Cardiac Catheterization , Cardiomyopathy, Dilated/drug therapy , Cardiomyopathy, Dilated/physiopathology , Cardiovascular Agents/therapeutic use , Ceftriaxone/administration & dosage , Drug Therapy, Combination , Echocardiography , Female , Humans , Infusions, Intravenous , Lyme Disease/drug therapy , Lyme Disease/physiopathology , Male , Middle Aged , Myocardial Contraction/drug effects , Stroke Volume/drug effects , Stroke Volume/physiology , Treatment Outcome , Ventricular Function, Left/drug effects , Ventricular Function, Left/physiology
9.
N Engl J Med ; 337(5): 289-94, 1997 Jul 31.
Article in English | MEDLINE | ID: mdl-9233865

ABSTRACT

BACKGROUND: Localized Lyme disease, manifested by erythema migrans, is usually treated with oral doxycycline or amoxicillin. Whether acute disseminated Borrelia burgdorferi infection should be treated differently from localized infection is unknown. METHODS: We conducted a prospective, open-label, randomized, multicenter study comparing parenteral ceftriaxone (2 g once daily for 14 days) with oral doxycycline (100 mg twice daily for 21 days) in patients with acute disseminated B. burgdorferi infection but without meningitis. The erythema migrans skin lesion was required for study entry, and disseminated disease had to be indicated by either multiple erythema migrans lesions or objective evidence of organ involvement. RESULTS: Of 140 patients enrolled, 133 had multiple erythema migrans lesions. Both treatments were highly effective. Rates of clinical cure at the last evaluation were similar among the patients treated with ceftriaxone (85 percent) and those treated with doxycycline (88 percent); treatment was considered to have failed in only one patient in each group. Among patients whose infections were cured, 18 of 67 patients in the ceftriaxone group (27 percent) reported one or more residual symptoms at the last follow-up visit, as did 10 of 71 patients in the doxycycline group (14 percent, P > or = 0.05). Mild arthralgia was the most common persistent symptom. Both regimens were well tolerated; only four patients (6 percent) in each group withdrew because of adverse events. CONCLUSIONS: In patients with acute disseminated Lyme disease but without meningitis, oral doxycycline and parenterally administered ceftriaxone were equally effective in preventing the late manifestations of disease.


Subject(s)
Anti-Bacterial Agents/therapeutic use , Ceftriaxone/therapeutic use , Cephalosporins/therapeutic use , Doxycycline/therapeutic use , Lyme Disease/drug therapy , Acute Disease , Administration, Oral , Adolescent , Adult , Aged , Aged, 80 and over , Anti-Bacterial Agents/adverse effects , Ceftriaxone/adverse effects , Cephalosporins/adverse effects , Child , Doxycycline/adverse effects , Female , Humans , Injections, Intramuscular , Injections, Intravenous , Male , Middle Aged , Prospective Studies , Treatment Outcome
10.
Ann Intern Med ; 124(9): 785-91, 1996 May 01.
Article in English | MEDLINE | ID: mdl-8610947

ABSTRACT

OBJECTIVE: To determine whether azithromycin or amoxicillin is more efficacious for the treatment of erythema migrans skin lesions, which are characteristic of Lyme disease. DESIGN: Randomized, double-blind, double-dummy, multicenter study. Acute manifestations and sequelae were assessed using a standardized format. Baseline clinical characteristics and response were correlated with serologic results. Patients were followed for 180 days. SETTING: 12 outpatient centers in eight states. PATIENTS: 246 adult patients with erythema migrans lesions at least 5 cm in diameter were enrolled and were stratified by the presence of flu-like symptoms (such as fever, chills, headache, malaise, fatigue, arthralgias, and myalgias) before randomization. INTERVENTION: Oral treatment with either amoxicillin, 500 mg three times daily for 20 days, or azithromycin, 500 mg once daily for 7 days. Patients who received azithromycin also received a dummy placebo so that the dosing schedules were identical. RESULTS: Of 217 evaluable patients, those treated with amoxicillin were significantly more likely than those treated with azithromycin to achieve complete resolution of disease at day 20, the end of therapy (88% compared with 76%; P=0.024). More azithromycin recipients (16%) than amoxicillin recipients (4%) had relapse (P=0.005). A partial response at day 20 was highly predictive of relapse (27% of partial responders had relapse compared with 6% of complete responders; P<0.001). For patients treated with azithromycin, development of an antibody response increased the possibility of achieving a complete response (81% of seropositive patients achieved a complete response compared with 60% of seronegative patients; P=0.043). Patients with multiple erythema migrans lesions were more likely than patients with single erythema migrans lesions (P<0.001) to have a positive antibody titer at baseline (63% compared with 17% for IgM; 39% compared with 16% for IgG). Fifty-seven percent of patients who had relapse were seronegative at the time of relapse. CONCLUSIONS: A 20-day course of amoxicillin was found to be an effective regimen for erythema migrans. Most patients were seronegative for Borrelia burgdorferi at the time of presentation with erythema migrans (65%) and at the time of relapse (57%).


Subject(s)
Amoxicillin/therapeutic use , Anti-Bacterial Agents/therapeutic use , Azithromycin/therapeutic use , Erythema Chronicum Migrans/drug therapy , Penicillins/therapeutic use , Adult , Amoxicillin/adverse effects , Anti-Bacterial Agents/adverse effects , Antibodies, Bacterial/blood , Azithromycin/adverse effects , Double-Blind Method , Enzyme-Linked Immunosorbent Assay , Erythema Chronicum Migrans/immunology , Female , Humans , Male , Penicillins/adverse effects , Prospective Studies , Recurrence , Treatment Failure
11.
Antimicrob Agents Chemother ; 40(2): 468-9, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8834900

ABSTRACT

Forty-one patients with erythema migrans were enrolled in an open-labelled pilot study of oral clarithromycin, 500 mg twice daily for 21 days, for the treatment of early Lyme disease. Immediately posttherapy, pretreatment signs and symptoms resolved among 91% of the 33 evaluable patients. At 6 months, all 28 of the evaluable patients were well. Clarithromycin shows promise as an effective agent for the treatment of early Lyme disease and warrants further study.


Subject(s)
Anti-Bacterial Agents/therapeutic use , Clarithromycin/therapeutic use , Lyme Disease/drug therapy , Adolescent , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged
12.
Clin Diagn Lab Immunol ; 1(4): 406-12, 1994 Jul.
Article in English | MEDLINE | ID: mdl-8556477

ABSTRACT

The major outer surface protein, OspA, of Borrelia burgdorferi is a lipoprotein which is a particular interest because of its potential as a vaccine candidate. However, serotypic and genetic analysis of OspA from both European and North American strains have demonstrated antigenic and structural heterogeneities. We purified OspA to homogeneity by exploiting its resistance to trypsin digestion. By treating spirochetes with trypsin and then using Triton X-114 extraction and ion-exchange chromatography, we obtained a yield of 2 mg of pure OspA protein per liter of culture. INtrinsic labeling with [14C]palmitic acid confirmed that OspA was lipidated, and partial digestion established lipidation at the amino-terminal end of the molecule. The reactivity of five anti-OspA murine monoclonal antibodies to nine different isolates of B. burgdorferi was ascertained by Western blot (immunoblot) analysis. Purified OspA was fragmented by enzymatic or chemical cleavage, and the monoclonal antibodies were able to define four distinct immunogenic domains. Further resolution of the epitope specificity to determine humoral and cellular immune responses to OspA has implications for vaccine development and for the utility of this protein as a reagent in diagnostic testing for Lyme borreliosis.


Subject(s)
Antigens, Surface/immunology , Bacterial Outer Membrane Proteins/immunology , Borrelia burgdorferi Group/immunology , Lipoproteins , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Antibody Specificity/immunology , Antigens, Surface/analysis , Antigens, Surface/isolation & purification , Bacterial Outer Membrane Proteins/analysis , Bacterial Outer Membrane Proteins/isolation & purification , Bacterial Vaccines , Binding Sites, Antibody , Blotting, Western , Borrelia burgdorferi Group/chemistry , Epitopes/immunology , Molecular Sequence Data , Palmitic Acid , Palmitic Acids , Peptide Mapping
13.
Int J Antimicrob Agents ; 3(4): 251-8, 1994.
Article in English | MEDLINE | ID: mdl-18611567
14.
Ann Intern Med ; 119(11): 1079-83, 1993 Dec 01.
Article in English | MEDLINE | ID: mdl-8239226

ABSTRACT

OBJECTIVE: To determine the prevalence and specificity of antibodies to Borrelia burgdorferi in patients with nonspirochetal subacute bacterial endocarditis and assess whether increased levels of antibodies to B. burgdorferi were attributable to rheumatoid factor. DESIGN: Retrospective case-control study. SETTING: Urban referral center in an area devoid of infected ticks as a source of endocarditis sera. PATIENTS: Sera from 30 consecutive patients with culture-proven subacute endocarditis between 1979 and 1981 were compared with 30 control sera collected between 1989 and 1990. In addition, sera from 20 consecutive patients with rheumatoid arthritis who were positive for rheumatoid factor were collected between 1991 and 1992. Sera were compared with a convenience sample from 15 patients who met the criteria for Lyme disease. MEASUREMENTS: Antibodies to B. burgdorferi were assessed by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. IgM rheumatoid factor was quantified using solid-phase radioimmunoassay or latex agglutination techniques. RESULTS: Thirteen of 30 patients with endocarditis (43%) compared with 3 of 30 normal controls (10%) had increased levels of antibodies to B. burgdorferi (P < 0.01). Of these 13 patients, only 1 had an immunoblot consistent with previous infection. The others had nonspecific immunoblots: 5 showed isolated 60-kd reactivity; 1 patient had isolated 41-kd reactivity; and 6 had no bands of reactivity. Immunoblots of the 3 controls with increased antibodies showed only isolated 41-kd reactivity. Thus, the specificity of the B. burgdorferi antibody test in patients with endocarditis was only 60% (95% CI, 42% to 78%), compared with 90% (CI, 79% to 100%) in controls. No correlation was noted between IgM rheumatoid factor and antibodies to B. burgdorferi in patients with endocarditis (r = 0.2; P > 0.2). Only 1 of 20 patients with rheumatoid arthritis without known bacterial infections had antibodies to B. burgdorferi. CONCLUSIONS: Although a positive ELISA test for B. burgdorferi may be a "true positive," a positive serologic test alone does not ensure that the clinical problem is due to Lyme borreliosis. Cross-reactive antibodies to shared epitopes between B. burgdorferi and the endocarditis organism may account for the high false-positive results.


Subject(s)
Antibodies, Bacterial/blood , Borrelia burgdorferi Group/immunology , Endocarditis, Bacterial/immunology , Endocarditis, Bacterial/microbiology , Cross Reactions , Diagnostic Errors , Enzyme-Linked Immunosorbent Assay , False Positive Reactions , Humans , Lyme Disease/diagnosis , Retrospective Studies , Rheumatoid Factor/blood
15.
Proc Natl Acad Sci U S A ; 90(21): 10163-7, 1993 Nov 01.
Article in English | MEDLINE | ID: mdl-8234271

ABSTRACT

The chromosomal genes fla and p93 and the ospA gene from a linear plasmid were sequenced from up to 15 isolates of Borrelia burgdorferi, which causes Lyme borreliosis in man. Comparison of the gene trees provides no evidence for genetic exchange between chromosomal genes, suggesting B. burgdorferi is strictly clonal. Comparison of the chromosomal gene trees with that of the plasmid-encoded ospA reveals that plasmid transfer between clones is rare. Evidence for intragenic recombination was found in only a single ospA allele. The analysis reveals three common clones and a number of rare clones that are so highly divergent that vaccines developed against one are unlikely to provide immunity to organisms from others. Consequently, an understanding of the geographic and genetic variability of B. burgdorferi will prove essential for the development of effective vaccines and programs for control. While the major clones might be regarded as different species, the clonal population structure, the geographic localization, and the widespread incidence of Lyme disease suggest that B. burgdorferi should remain the name for the entire array of organisms.


Subject(s)
Bacterial Vaccines , Borrelia burgdorferi Group/classification , Borrelia burgdorferi Group/genetics , Genes, Bacterial , Phylogeny , Base Sequence , Borrelia burgdorferi Group/immunology , Chromosomes, Bacterial , Cloning, Molecular , DNA Primers , Humans , Lyme Disease/immunology , Lyme Disease/microbiology , Molecular Sequence Data , Plasmids , Polymerase Chain Reaction
16.
Res Microbiol ; 144(4): 251-7, 1993 May.
Article in English | MEDLINE | ID: mdl-7504314

ABSTRACT

The p41 flagellin of Borrelia burgdorferi is the most common antigen recognized by serum of patients with Lyme borreliosis. This antigen shares amino acid homology, particularly in the amino and carboxy termini, with periflagellar antigens found in other microorganisms including Treponema pallidum. We cloned and expressed the p41 open reading frame in Escherichia coli and expressed it both as TrpE fusion and full-length unfused proteins. Also, we generated deletion constructs of various portions of the gene. Sera from patients with late Lyme borreliosis and secondary syphilis were used to identify the recombinant proteins by immunoblot analysis. Sera from 26 patients with Lyme borreliosis, 20 with secondary syphilis and 10 controls were used to identify cross-reactive domains of the B. burgdorferi flagellin. The variable region (amino acids 131-234) of the protein was recognized by 59% (15/26) of patients with late Lyme borreliosis compared to 30% (6/20) of patients with secondary syphilis and no (0/10) control patients. It appears that cross-reactive epitopes between B. burgdorferi and T. pallidum extend to the variable region of the flagellin.


Subject(s)
Borrelia burgdorferi Group/immunology , Flagellin/immunology , Lyme Disease/immunology , Syphilis/immunology , Borrelia burgdorferi Group/genetics , Cross Reactions , Electrophoresis, Polyacrylamide Gel , Epitopes/immunology , Escherichia coli/genetics , Flagellin/genetics , Humans , Immunoblotting , In Vitro Techniques , Open Reading Frames/genetics , Recombinant Proteins/analysis , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Treponema pallidum/immunology
17.
Infect Immun ; 60(10): 4309-21, 1992 Oct.
Article in English | MEDLINE | ID: mdl-1398941

ABSTRACT

Using immunoblots, we identified proteins of Borrelia burgdorferi recognized by sera from 62 patients with either acute or chronic Lyme disease. In all groups studied, the 41-kDa flagellar protein and a relatively minor 93-kDa protein (p93) were the most commonly recognized antigens in patients with acute and chronic disease due to B. burgdorferi. A murine monoclonal antibody (MAb 181.1) was developed against p93, and the antigen was detected by immunoblot analysis in four European and American strains of B. burgdorferi. On two-dimensional gel electrophoresis, p93 had an apparent pI of 6.8. Immunoelectronmicroscopy with MAb 181.1 demonstrated that p93 is located within the protoplasmic cylinder compartment of the organism. The gene encoding p93 was retrieved from a phage expression library. The derived amino acid sequence of p93 confirmed chemical characterization of the antigen, including its amino-terminal peptide sequence. The derived amino acid sequence predicted it to be predominantly alpha helical. A prominent antigenic domain located at the carboxy portion of the protein was recognized by human and rabbit polyclonal antisera and human (MAb D4) and mouse (MAb 181.1) MAbs.


Subject(s)
Antigens, Bacterial/analysis , Bacterial Proteins/analysis , Borrelia burgdorferi Group/immunology , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Base Sequence , Humans , Molecular Sequence Data
18.
Infect Immun ; 60(9): 3704-13, 1992 Sep.
Article in English | MEDLINE | ID: mdl-1379988

ABSTRACT

The gene encoding an immunoreactive Borrelia burgdorferi HSP70 homolog was isolated and characterized. The predicted amino acid sequence of this spirochetal protein confirms that this gene encodes a member of the HSP70 family of proteins. Although there appears to be a single copy of this gene on the spirochetal chromosome, two distinct transcripts hybridizing to the hsp70 probe are detected in RNA isolated from B. burgdorferi. The amount of spirochetal HSP70 RNA transcripts is shown to be thermally regulated. Antibodies in the serum of three Lyme arthritis patients and cloned T-cell lines isolated from one patient with Lyme arthritis recognize the expressed recombinant HSP70, indicating that it is an immunologically important spirochetal antigen. Antibodies in a rabbit antiserum, as well as antibodies in the serum of two of three Lyme arthritis patients examined, bound to expressed truncated recombinant HSP70s with 250 amino acids deleted from either the amino or carboxy terminus of the protein. However, antibodies in the serum of three Lyme arthritis patients, which were reactive with spirochetal HSP70, did not cross-react with human HSP70 proteins.


Subject(s)
Borrelia burgdorferi Group/immunology , Heat-Shock Proteins/immunology , Adolescent , Adult , Amino Acid Sequence , Animals , Antibodies, Bacterial/immunology , Base Sequence , DNA, Bacterial/chemistry , Female , Heat-Shock Proteins/genetics , Humans , Lyme Disease/immunology , Male , Molecular Sequence Data , RNA, Bacterial/analysis , Rabbits , T-Lymphocytes/immunology
19.
J Clin Microbiol ; 30(6): 1535-40, 1992 Jun.
Article in English | MEDLINE | ID: mdl-1378061

ABSTRACT

Purified flagellar protein (p41) of Borrelia burgdorferi (strain B31) was subjected to chemical cleavage with hydroxylamine or proteolysis with V8 protease, endoproteinase Asp-N, or alpha-chymotrypsin. The resulting polypeptides were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their positions in the published DNA sequence of the p41 protein were determined by amino-terminal sequencing and amino acid analysis. Epitope specificities of antibody binding by a monoclonal antibody raised by immunization of mice with purified flagella and pooled sera from patients with multiple erythema migrans, late Lyme borreliosis, or secondary syphilis were analyzed by Western blots (immunoblots) of peptides transferred to Immobilon polyvinylidene difluoride filters. The major epitope binding one murine monoclonal antibody (158) was localized to a carboxy-terminal domain that includes residues 300 to 336. The dominant epitopes binding human polyclonal antibodies are in the central portion of the molecule (residues 182 to 218) that is not conserved compared with other bacterial flagellins. Additional reactive epitopes were identified in the amino-terminal domain of the protein. Sera from patients with syphilis bound strongly to the amino-terminal conserved domain, providing a structural basis for cross-reactivity seen in standard enzyme-linked immunosorbent assays, but not to the central part of the molecule. Specific and cross-reactive antigenic determinants need to be considered in the design of improved immunodiagnostics for spirochetal diseases.


Subject(s)
Antigens, Bacterial/immunology , Borrelia burgdorferi Group/immunology , Flagella/immunology , Flagellin , Amino Acid Sequence , Antibodies, Bacterial/immunology , Antibodies, Monoclonal , Blotting, Western , Epitopes/immunology , Humans , Molecular Sequence Data , Peptide Mapping
20.
JAMA ; 267(10): 1364-7, 1992 Mar 11.
Article in English | MEDLINE | ID: mdl-1740859

ABSTRACT

OBJECTIVE: To determine central nervous system (CNS) involvement in acutely disseminated Borrelia burgdorferi infection by measurement of borrelia-specific DNA using the polymerase chain-reaction (PCR) assay and to compare the results of this with standard serological tests. DESIGN: Prospective study with laboratory investigators blinded to clinical data. SETTING: Multicenter office practice with a central reference laboratory. PATIENTS: Cerebrospinal fluid (CSF) was collected from 12 patients with acute disseminated Lyme borreliosis with less than 2 weeks of active disease. The normal control specimens came from 16 patients whose CSF samples had been sent to the clinical laboratory for tests unrelated to the present study. MAIN OUTCOME MEASURES: Clinical evidence of disease and laboratory abnormalities. RESULTS: Eight of the 12 patients (four of six with multiple areas of erythema migrans and four of six with cranial neuritis without erythema migrans) had B burgdorferi-specific DNA in their CSF. Among the 12 patients studied, nine had acute cranial neuritis and six had multiple erythema migrans lesions. Just four of the eight who were found to have spirochetal DNA in their CSF had complaints suggestive of CNS infection. In three of the PCR-positive CSF samples, no other abnormalities were noted. None of 16 samples from controls were positive in the PCR assay. CONCLUSION: B burgdorferi can invade the CNS early in the course of infection. Careful consideration should be given to choosing antibiotics that achieve adequate CSF levels in patients with disseminated infection.


Subject(s)
Borrelia burgdorferi Group/isolation & purification , Central Nervous System Diseases/microbiology , Lyme Disease/microbiology , Antibodies, Bacterial/analysis , Base Sequence , Borrelia burgdorferi Group/genetics , Borrelia burgdorferi Group/immunology , Central Nervous System Diseases/cerebrospinal fluid , Cerebrospinal Fluid/microbiology , DNA, Bacterial/analysis , Humans , Lyme Disease/cerebrospinal fluid , Molecular Sequence Data , Polymerase Chain Reaction , Prospective Studies
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