Barbara J. B. Johnson

Identification of a 47 kDa fibronectin‐binding protein expressed by Borrelia burgdorferi isolate B31

The attachment of pathogenic microorganisms to host cells and tissues is often mediated through the expression of surface receptors recognizing components of the extracellular matrix (ECM). Here, we investigate the ability of Borrelia spirochaetes to bind the ECM constituent, fibronectin. Borrelia lysates were separated by SDS–PAGE, transferred to nitrocellulose and probed with alkaline phosphatase‐labelled fibronectin (fibronectin‐AP). Five of six Borrelia species and four of eight B. burgdorferi sensu lato isolates expressed one or more fibronectin‐binding proteins. Borrelia burgdorferi isolate B31 expressed a 47 kDa (P47) fibronectin‐binding protein that was localized to the outer envelope based on susceptibility to proteinase K. The interaction of P47 with fibronectin was specific, and the region of fibronectin bound by P47 mapped to the gelatin/collagen binding domain. P47 was purified by affinity chromatography, digested with endoproteinase Lys‐C, and the peptide fragments analysed by liquid chromatography/tandem mass spectroscopy. A search of protein databases disclosed that the P47 peptide mass profile matched that predicted for the bbk32 gene product of B. burgdorferi isolate B31. The bbk32 gene was cloned into Escherichia coli, and the ability of recombinant BBK32 to bind fibronectin and inhibit the attachment of B. burgdorferi was demonstrated. The identification of BBK32 as a receptor for fibronectin binding may enhance our understanding of the pathogenesis and chronic nature of Lyme disease.

Serodiagnosis of Lyme Disease by Kinetic Enzyme-Linked Immunosorbent Assay Using Recombinant VlsE1 or Peptide Antigens of Borrelia burgdorferi Compared with 2-Tiered Testing Using Whole-Cell Lysates

Abstract In a study of US patients with Lyme disease, immunoglobulin (Ig) G and IgM antibody responses to recombinant Borrelia burgdorferi antigen VlsE1 (rVlsE1), IgG responses to a synthetic peptide homologous to a conserved internal sequence of VlsE (C6), and IgM responses to a synthetic peptide comprising the C-terminal 10 amino acid residues of a B. burgdorferi outer-surface protein C (pepC10) were evaluated by kinetic enzyme-linked immunoassay. At 99% specificity, the overall sensitivities for detecting IgG antibody to rVlsE1 or C6 in samples from patients with diverse manifestations of Lyme disease were equivalent to that of 2-tiered testing. When data were considered in parallel, 2 combinations (IgG responses to either rVlsE1 or C6 in parallel with IgM responses to pepC10) maintained high specificity (98%) and were significantly more sensitive than 2-tiered analysis in detecting antibodies to B. burgdorferi in patients with acute erythema migrans. In later stages of Lyme disease, the sensitivities of the in parallel tests and 2-tiered testing were high and statistically equivalent

Borrelia lonestari Infection after a Bite by an Amblyomma americanum Tick

Erythematous rashes that are suggestive of early Lyme disease have been associated with the bite of Amblyomma americanum ticks, particularly in the southern United States. However, Borrelia burgdorferi, the causative agent of Lyme disease, has not been cultured from skin biopsy specimens from these patients, and diagnostic serum antibodies usually have not been found. Borrelia lonestari sp nov, an uncultured spirochete, has been detected in A. americanum ticks by DNA amplification techniques, but its role in human illness is unknown. We observed erythema migrans in a patient with an attached A. americanum tick. DNA amplification of the flagellin gene flaB produced B. lonestari sequences from the skin of the patient that were identical to those found in the attached tick. B. lonestari is a probable cause of erythema migrans in humans.

Lyme Disease Testing by Large Commercial Laboratories in the United States

BACKGROUND Laboratory testing is helpful when evaluating patients with suspected Lyme disease (LD). A 2-tiered antibody testing approach is recommended, but single-tier and nonvalidated tests are also used. We conducted a survey of large commercial laboratories in the United States to assess laboratory practices. We used these data to estimate the cost of testing and number of infections among patients from whom specimens were submitted. METHODS Large commercial laboratories were asked to report the type and volume of testing conducted nationwide in 2008, as well as the percentage of positive tests for 4 LD-endemic states. The total direct cost of testing was calculated for each test type. These data and test-specific performance parameters available in published literature were used to estimate the number of infections among source patients. RESULTS Seven participating laboratories performed approximately 3.4 million LD tests on approximately 2.4 million specimens nationwide at an estimated cost of

Attenuating mutations in the E2 glycoprotein gene of Venezuelan equine encephalitis virus: Construction of single and multiple mutants in a full-length cDNA clone

492 million. Two-tiered testing accounted for at least 62% of assays performed; alternative testing accounted for <3% of assays. The estimated frequency of infection among patients from whom specimens were submitted ranged from 10% to 18.5%. Applied to the total numbers of specimens, this yielded an estimated 240 000 to 444 000 infected source patients in 2008. DISCUSSION LD testing is common and costly, with most testing in accordance with diagnostic recommendations. These results highlight the importance of considering clinical and exposure history when interpreting laboratory results for diagnostic and surveillance purposes.

Therapeutic efficacy and safety of chaperonin 10 in patients with rheumatoid arthritis: a double-blind randomised trial

Attenuated mutants of Venezuelan equine encephalitis virus (VEE) were isolated by selection for rapid penetration of cultured cells (R. E. Johnston and J. F. Smith, 1988, Virology 162, 437-443). Sequence analysis of these mutants identified candidate attenuating mutations at four loci in the VEE E2 glycoprotein gene: a double mutation at E2 codons 3 and 4, and single substitutions at E2 76, 120, and 209. Each candidate mutation was reproduced in an isogenic recombinant VEE strain using site-directed mutagenesis of a full-length cDNA clone of VEE. Characterization of these molecularly cloned mutant viruses showed that mutation at each of the four loci in the E2 gene was sufficient to confer both the accelerated penetration and attenuation phenotypes. Inoculation of the molecularly cloned viruses into rodent models that differ in their response to VEE suggested that individual mutations affected different aspects of VEE pathogenesis. Full-length clones containing multiple mutations were produced by combining independently attenuating mutations. Molecularly cloned viruses carrying two or three mutations were more attenuated in sensitive animal models than viruses which contained any single mutation alone. However, these highly attenuated strains still retained the ability to induce an immune response sufficient to protect against a high dose challenge with virulent VEE. These results indicate that production of a molecularly cloned live virus vaccine for VEE is feasible.

Duration of Tick Attachment as a Predictor of the Risk of Lyme Disease in an Area in which Lyme Disease Is Endemic

BACKGROUND Chaperonin 10 (heat shock protein 10, XToll) has anti-inflammatory properties related to the inhibition of Toll-like receptor signalling pathways. Our aim was to establish whether chaperonin 10 is safe and effective in the treatment of rheumatoid arthritis. METHODS In this randomised, double-blind, multicentre study, 23 patients with moderate to severe active rheumatoid arthritis receiving disease-modifying antirheumatic drugs were randomly allocated to three treatment groups receiving intravenous chaperonin 10 twice weekly for 12 weeks at doses of 5 mg (n=8), 7.5 mg (8), or 10 mg (7). The primary outcomes were change in disease activity score (DAS28) and improvement of core disease measures (American College of Rheumatology response score) from baseline to week 12. All analyses were done by intention to treat. This study is registered with the Australian Clinical Trials Registry, number ACTRNO12606000041550. FINDINGS Primary endpoint measures improved from day 14 in all groups and continued to improve to day 84. By end of study, a 20% improvement of core disease measures was seen in six (86%, 95% CI 43-100), a 50% improvement in four (57%, 14-86), and a 70% improvement in two (29%, 0-57) patients given the highest dose of chaperonin 10. Clinical remission (as defined by a DAS28 <2.6) was achieved in three (13%) of 23 patients. Three individuals dropped out during the study: one in the 5 mg group (rheumatoid arthritis not controlled), one in the 7.5 mg group (adverse event), and one in the 10 mg group (lost to follow-up). The most common adverse events were exacerbation of rheumatoid arthritis (both during and after the study) and upper respiratory tract infection. Only one adverse event was judged to be of severe intensity. INTERPRETATION Chaperonin 10 seems to be well tolerated and efficacious in treatment of the symptoms of rheumatoid arthritis, at least in the short term.

Resistance to Lyme disease in decorin-deficient mice

Animal studies have shown an exponential increase in the risk of Borrelia burgdorferi infection after 48-72 h of deer tick attachment. Persons with tick bites were prospectively studied to determine if those with prolonged tick attachment constitute a high-risk group for infection. Ticks were identified, measured for engorgement, and assayed by polymerase chain reaction (PCR) for B. burgdorferi DNA. Duration of attachment was determined from the scutal index of engorgement. Of 316 submissions, 229 were deer ticks; 14% were positive by PCR. Paired sera and an intact tick for determination of duration of attachment were available for 105 subjects (109 bites). There were 4 human cases (3.7% of bites) of B. burgdorferi infection. The incidence was significantly higher for duration of attachment > or =72 h than for <72 h: 3 (20%) of 15 vs. 1 (1.1%) of 94 (P = .008; odds ratio, 23.3; 95% confidence interval, 2.2-242). PCR was an unreliable predictor of infection. Tick identification and measurement of engorgement can be used to identify a small, high-risk subset of persons who may benefit from antibiotic prophylaxis.

Single-tier testing with the C6 peptide ELISA kit compared with two-tier testing for Lyme disease

Microbial adhesion to the host tissue represents an early, critical step in the pathogenesis of most infectious diseases. BORRELIA: burgdorferi, the causative agent of Lyme disease (LD), expresses two surface-exposed decorin-binding adhesins, DbpA and DbpB. A decorin-deficient (Dcn(-/-)) mouse was recently developed and found to have a relatively mild phenotype. We have now examined the process of experimental LD in Dcn(-/-) mice using both needle inoculation and tick transmission of spirochetes. When exposed to low doses of the infective agent, Dcn(-/-) mice had fewer Borrelia-positive cultures from most tissues analyzed than did Dcn(+/+) or Dcn(+/-) mice. When the infection dose was increased, similar differences were not observed in most tissues but were seen in bacterial colonization of joints and the extent of Borreila-induced arthritis. Quantitative PCR demonstrated that joints harvested from Dcn(-/-) mice had diminished Borrelia numbers compared with issues harvested from Dcn(+/+) controls. Histological examination also revealed a low incidence and severity of arthritis in Dcn(-/-) mice. Conversely, no differences in the numbers of Borreila-positive skin cultures were observed among the different genotypes regardless of the infection dose. These differences, which were observed regardless of genetic background of the mice (BALB/c or C3H/HeN) or method of infection, demonstrate the importance of decorin in the pathogenesis of LD.

Mapping the Ligand-Binding Region of Borrelia burgdorferi Fibronectin-Binding Protein BBK32

For the diagnosis of Lyme disease, the 2-tier serologic testing protocol for Lyme disease has a number of shortcomings including low sensitivity in early disease; increased cost, time, and labor; and subjectivity in the interpretation of immunoblots. In this study, the diagnostic accuracy of a single-tier commercial C6 ELISA kit was compared with 2-tier testing. The results showed that the C6 ELISA was significantly more sensitive than 2-tier testing with sensitivities of 66.5% (95% confidence interval [CI] 61.7-71.1) and 35.2% (95% CI 30.6-40.1), respectively (P < 0.001) in 403 sera from patients with erythema migrans. The C6 ELISA had sensitivity statistically comparable to 2-tier testing in sera from Lyme disease patients with early neurologic manifestations (88.6% versus 77.3%, P = 0.13) or arthritis (98.3% versus 95.6%, P = 0.38). The specificities of C6 ELISA and 2-tier testing in over 2200 blood donors, patients with other conditions, and Lyme disease vaccine recipients were found to be 98.9% and 99.5%, respectively (P < 0.05, 95% CI surrounding the 0.6 percentage point difference of 0.04 to 1.15). In conclusion, using a reference standard of 2-tier testing, the C6 ELISA as a single-step serodiagnostic test provided increased sensitivity in early Lyme disease with comparable sensitivity in later manifestations of Lyme disease. The C6 ELISA had slightly decreased specificity. Future studies should evaluate the performance of the C6 ELISA compared with 2-tier testing in routine clinical practice.