Immunodiagnostic tests for Lyme disease.

Standardized serologic tests for Lyme disease are needed, as isolation or in situ demonstration of the spirochete has proved difficult. At the Centers for Disease Control (CDC), an indirect immunofluorescence assay (IFA) was modified from a previously described IFA, and an enzyme-linked immunosorbent assay (ELISA) was developed with soluble spirochetal antigens. Both tests were evaluated with sera from Lyme disease patients, normal controls, and patients with other diseases. They were highly specific for Lyme disease when sera from patients with syphilis were excluded. Sensitivity varied with disease stage: for patients with erythema chronicum migrans alone, the IFA was 53 percent sensitive and the ELISA was 67 percent sensitive. In contrast, all patients with complicated Lyme disease had at least one serum specimen positive in both tests. Twenty-six percent of the sera from 289 patients with suspected Lyme disease that were submitted to CDC in 1983 had IFA titers greater than or equal to 256 and thus were considered positive. Both tests should be useful diagnostic and epidemiologic aids.     

Teratogen update: Lyme disease.

We reviewed the world literature concerning the reproductive effects of Lyme disease (LD). Borrelia burgdorferi, which is the etiology of LD, is a spirochete and, as such, may share the potential for causing fetal infection, which may occur in the setting of maternal spirochetemia. Information concerning the effects of gestational LD derives from case reports and series, epidemiologic studies, and experimental animal models. Although provocative, these studies fail to define a characteristic teratogenic effect. However, skin and cardiac involvement have predominated in some reports. Pregnancy wastage has been suggested primarily by animal studies. Gestational LD appears to be associated with a low risk of adverse pregnancy outcome, particularly with appropriated antibiotic therapy. Suggestions for management of clinical situations are presented.     

The antibody response in Lyme disease.

We determined the antibody response against the Ixodes dammini spirochete in Lyme disease patients by indirect immunofluorescence and an enzyme-linked immunosorbent assay (ELISA). The specific IgM response became maximal three to six weeks after disease onset, and then declined, although titers sometimes remained elevated during later disease. Specific IgM levels correlated directly with total serum IgM. The specific IgG response, often delayed initially, was nearly always present during neuritis and arthritis, and frequently remained elevated after months of remission. Although results obtained by indirect immunofluorescence and the ELISA were similar, the ELISA was more sensitive and specific. Cross-reactive antibodies from patients with other spirochetal infections were blocked by absorption of sera with Borrelia hermsii, but titers of Lyme disease sera were also decreased. To further characterize the specificity of the humoral immune response against the I. dammini spirochete, 35S-methionine-labeled spirochetal antigens were identified by immunoprecipitation with sera from Lyme arthritis patients. These polypeptides had molecular weights of 62, 60, 47, 37, 22, 18, and 15 kDa, and were not recognized by control sera. We conclude that the ELISA, without absorption, is the best method to assay the humoral immune response in Lyme disease, and we have identified methionine-containing spirochetal polypeptides that may be important in Lyme arthritis.     

DNA characterization of Lyme disease spirochetes.

Lyme disease spirochetes (LDS) have phenotypic characteristics of both treponemes and borreliae. To ascertain whether one or more species of LDS exist, as well as their taxonomic status, we determined the DNA base (G + C) content for three strains of LDS, the DNA relatedness of ten strains isolated in the United States or Europe, and the DNA relatedness of LDS to other spirochetes. The G + C content of the three LDS strains was 28.1-29.0 mol%, most similar to those of Borellia hermsii (30.6 mol %) and Treponema hyodysenteriae (25.6 mol %) among the other spirochetes tested. DNA hybridization studies of nine LDS strains to a reference strain isolated from human blood revealed divergence (unpaired bases) within related nucleotide sequences of only 0.0-1.0 percent, indicating the strains were one species. Similarly, relatedness values of seven strains to the reference strain were high: 58-98 percent (mean, 71 percent) in 50 degrees C reactions and 50-93 percent (mean, 69 percent) in 65 degrees C reactions. Labeled DNA from B. hermsii was 30-40 percent related to three Lyme disease spirochete strains in 50 degrees C reactions and 8-10 percent related in 65 degrees C reactions. In contrast, DNA from the reference LDS strain showed relatedness of only 1 percent to DNAs of two leptospires and only 16 percent to DNA from T. hyodysenteriae. We conclude that LDS are a single species, genetically unlike treponemes or leptospires, which belong in the genus Borrelia.     

Taxonomy of the Lyme disease spirochetes.

Morphology, physiology, and DNA nucleotide composition of Lyme disease spirochetes, Borrelia, Treponema, and Leptospira were compared. Morphologically, Lyme disease spirochetes resemble Borrelia. They lack cytoplasmic tubules present in Treponema, and have more than one periplasmic flagellum per cell end and lack the tight coiling which are characteristic of Leptospira. Lyme disease spirochetes are also similar to Borrelia in being microaerophilic, catalase-negative bacteria. They utilize carbohydrates such as glucose as their major carbon and energy sources and produce lactic acid. Long-chain fatty acids are not degraded but are incorporated unaltered into cellular lipids. The diamino amino acid present in the peptidoglycan is ornithine. The mole % guanine plus cytosine values for Lyme disease spirochete DNA were 27.3-30.5 percent. These values are similar to the 28.0-30.5 percent for the Borrelia but differed from the values of 35.3-53 percent for Treponema and Leptospira. DNA reannealing studies demonstrated that Lyme disease spirochetes represent a new species of Borrelia, exhibiting a 31-59 percent DNA homology with the three species of North American borreliae. In addition, these studies showed that the three Lyme disease spirochetes comprise a single species with DNA homologies ranging from 76-100 percent. The three North American borreliae also constitute a single species, displaying DNA homologies of 75-95 percent. Lyme disease spirochetes and Borrelia exhibited little or no DNA homology (0-2 percent) with the Treponema or Leptospira. Plasmids were present in the three Lyme disease spirochetes and the three North American borreliae.     

Lyme disease in northwestern coastal California.

To determine the incidence of physician-diagnosed Lyme disease in an endemic area of California, an active surveillance program was implemented in Lake, Mendocino, Sonoma, and southern Humboldt counties. More than 200 medical care providers were called monthly for their list of suspected cases of Lyme disease. Pertinent information was abstracted from the medical record of each patient. Of 153 cases of possible early Lyme disease ascertained from July 1991 to December 1992, 37% consisted of physician-diagnosed erythema migrans. Only 58% of erythema migrans rashes were at least 5 cm in diameter. An additional 43 patients had suspicious rashes not classified as erythema migrans. Of 166 patients with possible late-stage Lyme disease, 31% had specific clinical symptoms and 75% had a positive serologic test. With an incident case defined as physician-diagnosed erythema migrans of at least 5 cm in diameter, the annual incidence of Lyme disease in northwestern coastal California according to active surveillance only was 5.5 per 100,000. The rate of Lyme disease in California is substantially lower than that in the Atlantic northeastern United States. Many suspected cases of Lyme disease in this endemic area do not meet surveillance criteria, which are intentionally restrictive. Although some of the illnesses not meeting surveillance criteria may be due to infection with Borrelia burgdorferi, it appears that Lyme disease is being overdiagnosed in this area.     

Recovery of Lyme disease spirochetes from patients.

Since the summer of 1982, we have cultured patient specimens for Lyme disease spirochetes. Of 118 patients cultured, four specimens yielded spirochetes: two from blood, one from a skin biopsy specimen of erythema chronicum migrans (ECM), and one from cerebrospinal fluid. All four isolates appeared identical when examined with a monoclonal antibody. However, attempts to recover the spirochete from synovium or synovial fluid were unsuccessful. In addition, the organism could not be visualized in skin or synovial biopsy specimens using the avidin-biotin peroxidase complex detection system. Thus, the current yield in culturing spirochetes from patients is quite low, and it is not yet known whether the organism is still alive later in the disease when arthritis is present.     

Telomere resolution in the Lyme disease spirochete.

The genus Borrelia includes the causative agents of Lyme disease and relapsing fever. An unusual feature of these bacteria is a genome that includes linear DNA molecules with covalently closed hairpin ends referred to as telomeres. We have investigated the mechanism by which the hairpin telomeres are processed during replication. A synthetic 140 bp sequence having the predicted structure of a replicated telomere was shown to function as a viable substrate for telomere resolution in vivo, and was sufficient to convert a circular replicon to a linear form. Our results suggest that the final step in the replication of linear Borrelia replicons is a site-specific DNA breakage and reunion event to regenerate covalently closed hairpin ends. The telomere substrate described here will be valuable both for in vivo manipulation of linear DNA in Borrelia and for in vitro studies to identify and characterize the telomere resolvase.     

Progressive hemifacial atrophy associated with Lyme disease.

A case is described of progressive hemifacial atrophy occurring in a child with Lyme disease. Borrelial infection may have been an etiologic factor in the progressive hemifacial atrophy, which was ultimately treated with an SIEA free-flap transfer.