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Chronic Lyme Disease: a Persistent Inflammatory Infection

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The potential for transport and dissemination of certain pathogenic microorganisms by migratory birds is of concern. Migratory birds might be involved in dispersal of microorganisms as their biological carriers. mechanical carriers. or as carriers of infected hematophagous ver-parasites (e.g., ixodid ticks). Many species of microorganisms pathogenic to homeothermic e vertebrates including humans have been associated with free-living migratory birds. Migratory birds of diverse species can play significant roles in the ecology and circulation of some arboviruses (e.g., eastern and western equine encephalomyelitis and Sindbis alphaviruses. West Nile and St., Louis encephalitis flaviviruses), influenza A virus. Newcastle disease virus, duck plague herpes-virus, Chlamydophila psittaci, Anaplasma phagocytophilum. Borrelia burgdorferi sensu lato. Campylobacter, jejuni, Salmonella enterica, Pasteurella multocida, Mycobacterium avium, Candida spp., and avian hematozoans. The efficiency of dispersal of pathogenic microorganisms depends on a wide variety of biotic and abiotic factors affecting the survival or the agent in. or disappearance from, a habitat or ecosystem in a new geographic area.
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Borrelia burgdorferi sensu lato, the spirochete that causes human Lyme borreliosis (LB), is a genetically and phenotypically divergent species. In the past several years, various molecular approaches have been developed and used to determine the phenotypic and genetic heterogeneity within the LB-related spirochetes and their potential association with distinct clinical syndromes. These methods include serotyping, multilocus enzyme electrophoresis, DNA-DNA reassociation analysis, rRNA gene restriction analysis (ribotyping), pulsed-field gel electrophoresis, plasmid fingerprinting, randomly amplified polymorphic DNA fingerprinting analysis, species-specific PCR and PCR-based restriction fragment length polymorphism (RFLP) analysis, and sequence analysis of 16S rRNA and other conserved genes. On the basis of DNA-DNA reassociation analysis, 10 different Borrelia species have been described within the B. burgdorferi sensu lato complex: B. burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, Borrelia japonica, Borrelia andersonii, Borrelia valaisiana, Borrelia lusitaniae, Borrelia tanukii, Borrelia turdi, and Borrelia bissettii sp. nov. To date, only B. burgdorferi sensu stricto, B. garinii, and B. afzelii are well known to be responsible for causing human disease. Different Borrelia species have been associated with distinct clinical manifestations of LB. In addition, Borrelia species are differentially distributed worldwide and may be maintained through different transmission cycles in nature. In this paper, the molecular methods used for typing of B. burgdorferi sensu lato are reviewed. The current taxonomic status of B. burgdorferi sensu lato and its epidemiological and clinical implications, especially correlation between the variable clinical presentations and the infecting Borrelia species, are discussed in detail.
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Lyme borreliosis (Lyme disease), a tick-borne spirochetal illness, has multisystemic involvement and is rapidly increasing in certain areas of the United States. Although its neurologic manifestations are becoming increasingly well recognized, its psychiatric presentations are not well known. The first section of this paper will provide an overview of Lyme borreliosis and a review of the relevant neuropsychiatric literature. The second section will provide clinical descriptions of some common neuropsychiatric symptoms as well as a discussion of the problems typically faced by patients with this illness. Guidelines to assist the clinician in working with these patients will be presented.
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The significance of various antibodies against Borrelia burgdorferi was studied by Western blot (immunoblot) by using 578 human serum samples. The proteins regularly detected by using samples from patients with Lyme borreliosis were those with bands with molecular masses of 94, 83, 75, 66, 60, 55, 46, 41, 39, 34, 31, 29, 22, and 17 kDa. The detectable frequencies of most of these proteins appeared to be significantly different between the sera from patients with Lyme borreliosis and those from normal control individuals as well as from the group with syphilis. The 39-kDa protein band recognized by polyvalent antibody was found to be the most significant marker for Lyme borreliosis. Furthermore, an anti-39-kDa immunoglobulin M response was detected in the samples from patients with early-stage Lyme borreliosis. Results from the use of monoclonal antibodies and patient sera revealed that the 39- and 41-kDa proteins may be structurally related but are immunologically distinct antigens. The significance of antibody reactivities to the 41-, 94-, 22-, 31-, and 34-kDa protein bands is also discussed.
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Using immunoblots, we identified proteins of Borrelia burgdorferi bound by IgM and IgG antibodies during Lyme disease. In 12 patients with early disease alone, both the IgM and IgG responses were restricted primarily to a 41-kD antigen. This limited response disappeared within several months. In contrast, among six patients with prolonged illness, the IgM response to the 41-kD protein sometimes persisted for months to years, and late in the illness during arthritis, a new IgM response sometimes developed to a 34-kD component of the organism. The IgG response in these patients appeared in a characteristic sequential pattern over months to years to as many as 11 spirochetal antigens. The appearance of a new IgM response and the expansion of the IgG response late in the illness, and the lack of such responses in patients with early disease alone, suggest that B. burgdorferi remains alive throughout the illness.
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The sensitivities and specificities of three enzyme-linked immunosorbent assays (ELISAs) for Borrelia burgdorferi antibodies were compared for 41 patients presenting with symptoms compatible with late Lyme borreliosis (LB) and 37 healthy controls. All subjects were living in southwestern Finland, where LB is endemic. Only patients with culture- or PCR-proven disease were enrolled in the study. The antigens of the ELISAs consisted of sonicated spirochetes, 41-kDa flagellin, and recombinant P39 protein of B. burgdorferi. Fifteen patients had strongly or moderately positive results in the serological assay(s), 19 patients had only weakly positive or borderline antibody levels, and the remaining 7 patients were seronegative by ELISA. The sensitivities of the ELISAs were 78.0% with sonicate antigen, 41.5% with 41-kDa flagellin, and 14.6% with P39 protein. The specificities of the tests were 89.2, 86.5, and 94.6%, respectively. The sonicate antigen ELISA seems to be an effective screening method. These results show that antibodies to B. burgdorferi may be present in low levels or even absent in patients with culture- or PCR-proven late LB. Therefore, in addition to serological testing, the use of PCR and cultivation is recommended in the diagnosis of LB.
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A 40 year old man presented with progressive personality changes in the previous six months. Specific serological tests for syphilis in blood and CSF were highly positive and CSF sedimentation showed signs of an inflammatory process. Ten hours after the start of penicillin treatment a severe symptomatic Jarisch-Herxheimer reaction with alteration of level of consciousness, pupillary changes, and focal neurological signs developed. Jarisch-Herxheimer reaction may occur in various settings, particularly in the treatment of syphilis. Investigation of CSF before the treatment may predict a potential risk. Corticosteroid treatment has been suggested for prevention.
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In October 1994, the Second National Conference on the Serologic Diagnosis of Lyme Disease recommended a two-step approach to serological testing. The first step was the performance of an enzyme-linked immunosorbent assay (ELISA); the second step was a confirmatory immunoblot. New criteria for the interpretation of a positive immunoblot were also recommended. The committee decided to omit the 31- and 34-kDa bands (OspA and OspB, respectively) from the choice of bands considered diagnostic for a positive immunoblot. Since we had previously included these in our diagnostic criteria for Lyme disease-positive immunoblots, we reviewed data for all patients attending a Lyme disease center with positive ELISAs and immunoblot assays for Lyme disease from 1 September 1992 to 31 December 1993. The criteria for a positive Western blot (immunoblot) were the presence of 5 or 12 bands, including the 10 recommended by the conference, and the presence of the 31- and 34-kDa protein bands. Of the 136 patients evaluated, 50 were considered to have Lyme disease. Of these 50, 4 (8%) would not have met immunoblot criteria for the diagnosis if the new recommendations were used. Had the 31- and 34-kDa bands been included as part of the diagnostic requirements for immunoblot, these patients would have been included. Although overdiagnosis of Lyme disease appears to be the more frequent problem, our concern is that the exclusion of the 31- and 34-kDa protein bands from the diagnostic criteria may result in the underdiagnosis of Lyme disease by those who would rely too heavily on serological confirmation. The addition of the 31- and 34-kDa bands to those recommended for confirmatory immunoblot should be reconsidered.
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As clinical persistence of Borrelia burgdorferi in patients with active Lyme borreliosis occurs despite obviously adequate antibiotic therapy, in vitro investigations of morphological variants and atypical forms of B. burgdorferi were undertaken. In an attempt to learn more about the variation of B. burgdorferi and the role of atypical forms in Lyme borreliosis, borreliae isolated from antibiotically treated and untreated patients with the clinical diagnosis of definite and probable Lyme borreliosis and from patient specimens contaminated with bacteria were investigated. Furthermore, the degeneration of the isolates during exposure to penicillin G in vitro was analysed. Morphological analysis by darkfield microscopy and scanning electron microscopy revealed diverse alterations. Persisters isolated from a great number of patients (60-80%) after treatment with antibiotics had an atypical form. The morphological alterations in culture with penicillin G developed gradually and increased with duration of incubation. Pleomorphism, the presence of elongated forms and spherical structures, the inability of cells to replicate, the long period of adaptation to growth in MKP-medium and the mycoplasma-like colonies after growth in solid medium (PMR agar) suggest that B. burgdorferi produce spheroplast-L-form variants. With regard to the polyphasic course of Lyme borreliosis, these forms without cell walls can be a possible reason why Borrelia survive in the organism for a long time (probably with all beta-lactam antibiotics) [corrected] and the cell-wall-dependent antibody titers disappear and emerge after reversion.
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Recent guidelines established by the Association of State and Territorial Public Health Laboratory Directors (ASTPHLD) and the U.S. Centers for Disease Control and Prevention (CDC) recommend the use of a two-test protocol for the serologic diagnosis of Lyme disease (LD). The two-test protocol relies on a sensitive screening test, which is followed by specific immunoglobulin M (IgM) and/or IgG immunoblotting (IB), depending on the date of disease onset, of all samples with equivocal and positive screening test results. We evaluated a commercially available IgM-IgG enzyme-linked immunosorbent assay (ELISA) and separate IB tests for IgM and IgG antibodies to Borrelia burgdorferi as candidate assays for the two-test protocol. Serum samples obtained from healthy controls (n = 29), from patients with diagnoses or laboratory findings associated with serologic cross-reactivity to LD (n = 24), and from patients with well-documented early- and late-stage LD provided by the CDC and the College of American Pathologists (n = 53) were examined to determine each assay's individual sensitivity and specificity. No false-positive results were detected among the healthy controls by either ELISA or IB, whereas four false-positive ELISA results were recorded within the cross-reactive group. None of these sera, however, were positive for either IgM or IgG reactivity according to IB band criteria. With regard to the patients with LD, we determined the sensitivity and specificity of the ELISA to be 96 and 100%, respectively, compared with the reference data provided for these specimens. When we compared our IB results with data from CDC, the assay sensitivity and specificity were 80 and 96.2%, respectively, for IgM and 81.8 and 95.8%, respectively, for IgG. Pursuant to this evaluation we assessed the suitability of the two-test protocol by performing a retrospective analysis using clinical history to define samples as positive or negative for LD. We determined clinical sensitivity and specificity for all study subjects (n = 112) to be 50 and 100%, respectively. A reduction in the clinical sensitivity of the two-test protocol was associated with a lack of antibody response or seroconversion in LD patients treated with antibiotics. We conclude that the CDC-ASTPHLD guidelines provide useful criteria for test performance and interpretation aimed at standardizing the serologic diagnosis of LD.
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We have identified and characterized an elaborate genetic system in the Lyme disease spirochete Borrelia burgdorferi that promotes extensive antigenic variation of a surface-exposed lipoprotein, VlsE. A 28 kb linear plasmid of B. burgdorferi B31 (lp28-1) was found to contain a vmp-like sequence (vls) locus that closely resembles the variable major protein (vmp) system for antigenic variation of relapsing fever organisms. Portions of several of the 15 nonexpressed (silent) vls cassette sequences located upstream of vlsE recombined into the central vlsE cassette region during infection of C3H/HeN mice, resulting in antigenic variation of the expressed lipoprotein. This combinatorial variation could potentially produce millions of antigenic variants in the mammalian host.
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The Centers for Disease Control and Prevention (CDC) recommend a two-test approach for the serodiagnosis of Lyme disease (LD), with EIA testing followed by Western immunoblotting (WB) of EIA-equivocal and -positive specimens. This approach was compared with a simplified two-test approach (WB of EIA equivocals only) and WB alone for early LD. Case-patients with erythema migrans (EM) rash 3⩾5 cm were recruited from three primary-care practices in LD-endemic areas to provide acute-(S1) and convalescent-phase serum specimens (S2). The simplified approach had the highest sensitivity when either S1 or S2 samples were tested, nearly doubling when S2 were tested, while decreasing slightly for the other two approaches. Accordingly, the simplified approach had the lowest negative likelihood ratio for either S1 or S2. For early LD with EM, the simplified approach performed well and was less costly than the other testing approaches since less WB is required.
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It has been demonstrated previously that motile Borrelia burgdorferi cells transform into non-motile cyst-forms when incubated for several weeks in BSKII (a complex medium) lacking rabbit serum. B. burgdorferi cells cannot synthesize fatty acids de novo and serum is thought to provide a source of fatty acids and lipids. When B. burgdorferi cells were serum-starved in defined RPMI medium, -90% of the cells formed spherical cysts within 48 h. Cyst formation was inhibited by tetracycline. Cyst opening and recovery of vegetative cells was rapidly induced by the addition of either BSKII or rabbit serum. The percentage of viable cells recovered from cysts ranged from 2.9% to 52-5%. Viability was inversely proportional to cyst age. Protein synthesis by B. burgdorferi during serum starvation was examined by labelling cells with Tran35S-Label and analysing the labelled proteins by two-dimensional gel electrophoresis and fluorography. The synthesis of over 20 proteins was induced during serum starvation. Western blots of proteins from vegetative cells and cysts probed with sera from either B. burgdorferi-infected humans or monkeys revealed that several cyst proteins were antigenic. These data suggest that cells of B. burgdorferi, although possessing a small genome and extremely limited biosynthetic capabilities, rapidly respond to conditions of serum starvation by inducing changes in protein synthesis and cell morphology. This study may help explain how cells of B. burgdorferi can survive periods of nutrient deprivation in different hosts and host tissues.
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Several pathogens of humans and domestic animals depend on hematophagous arthropods to transmit them from one vertebrate reservoir host to another and maintain them in an environment. These pathogens use antigenic variation to prolong their circulation in the blood and thus increase the likelihood of transmission. By convergent evolution, bacterial and protozoal vector-borne pathogens have acquired similar genetic mechanisms for successful antigenic variation. Borrelia spp. and Anaplasma marginale (among bacteria) and African trypanosomes, Plasmodium falciparum, and Babesia bovis (among parasites) are examples of pathogens using these mechanisms. Antigenic variation poses a challenge in the development of vaccines against vector-borne pathogens.
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To document the persistence of Borrelia burgdorferi in ligamentous tissue samples obtained from a woman with chronic Lyme borreliosis. Spirochetes were isolated from samples of ligamentous tissue, and the spirochetes were characterized antigenetically and by molecular biology techniques. The ligamentous tissue was examined by electron microscopy. Humoral and cellular immune responses were analyzed. Choroiditis was the first recognized manifestation of Lyme disease in this patient. Despite antibiotic therapy, there was progression to a chronic stage, with multisystem manifestations. The initially significant immune system activation was followed by a loss of the specific humoral immune response and a decrease in the cellular immune response to B burgdorferi over the course of the disease. "Trigger finger" developed, and a portion of the flexor retinaculum obtained at surgery was cultured. Viable spirochetes were identified. Ultramorphologically, the spirochetes were situated between collagen fibers and along fibroblasts, some of which were deeply invaginated by these organisms. The cultured bacteria were identified as B burgdorferi by reactions with specific immune sera and monoclonal antibodies, and by polymerase chain reaction amplification and Southern blot hybridization techniques. To our knowledge, this is the first report of the isolation of B burgdorferi from ligamentous tissue. This suggests that tendon tissues serve as a specific site of spirochete residence in human hosts.
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To investigate the role of B. henselae in patients with symptoms suggesting neuroborreliosis, serum and cerebrospinal fluid samples were tested with serological and PCR methods. Among 17 examined patients, in 12 cases Borrelia burgdorferi infections were detected, in 1 case Bartonella henselae infection was ascertained, and in two patients mixed B. burgdorferi and B. henselae infections were found. These results indicate that mixed infections should be taken into consideration in establishing diagnosis of neurological disorders. Further study of this conclusion is needed.
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The Lyme disease-associated spirochete, Borrelia burgdorferi, is maintained in enzootic cycles involving Ixodes ticks and small mammals. Previous studies demonstrated that B. burgdorferi expresses outer surface protein A (OspA) but not OspC when residing in the midgut of unfed ticks. However, after ticks feed on blood, some spirochetes stop making OspA and express OspC. Our current work examined the timing and frequency of OspA and OspC expression by B. burgdorferi in infected Ixodes scapularis nymphs as they fed on uninfected mice and in uninfected I. scapularis larvae and nymphs as they first acquired spirochetes from infected mice. Smears of midguts from previously infected ticks were prepared at 12- or 24-h intervals following attachment through repletion at 96 h, and spirochetes were stained for immunofluorescence for detection of antibodies to OspA and OspC. As shown previously, prior to feeding spirochetes in nymphs expressed OspA but not OspC. During nymphal feeding, however, the proportion of spirochetes expressing OspA decreased, while spirochetes expressing OspC became detectable. In fact, spirochetes rapidly began to express OspC, with the greatest proportion of spirochetes having this protein at 48 h of attachment and then with the proportion decreasing significantly by the time that the ticks had completed feeding. In vitro cultivation of the spirochete at different temperatures showed OspC to be most abundant when the spirochetes were grown at 37 degrees C. Yet, the synthesis of this protein waned with continuous passage at this temperature. Immunofluorescence staining of spirochetes in smears of midguts from larvae and nymphs still attached or having completed feeding on infected mice demonstrated that OspA but not OspC was produced by these spirochetes recently acquired from mice. Therefore, the temporal synthesis of OspC by spirochetes only in feeding ticks that were infected prior to the blood meal suggests that this surface protein is involved in transmission from tick to mammal but not from mammal to tick.
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Forty-eight patients with erythema chronicum migrans (ECM) were studied prospectively for 6 to 18 months. Twenty-six patients had no later symptoms, but 22 subsequently developed Lyme arthritis and 9 of them also experienced neurologic abnormalities. Eighty-seven percent of patients with active ECM followed by subsequent involvement had cryoglobulins containing IgM compared to only 13% of those with active ECM and no later symptoms. The former group also had significantly lower IgG, C3 and C4 levels. Sixty-seven percent of patients still had serum cryoglobulins when neurologic disease was most active, and 45% had them when joint symptoms were most severe, but only 11% continued to have small amounts in remission. The number of patients who continued to have serum cryoglobulins with recurrent attacks of arthritis decreased with time. In contrast, patients always had cryoglobulins in joint fluid, a finding Lyme arthritis shares with rheumatoid arthritis. The cryoprecipitates from 2 of 10 patients contained particles with internal structure, but their viral nature is problematic. All components of antisera obtained from goats and rabbits immunized with cryoglobulins were absorbed by normal human sera. The amount of IgM in cryoglobulins correlated directly with serum IgM, which generally rose during exacerbations and fell during remissions; serum IgG and IgA moved conversely. Thus, IgM was an important correlate of clinical disease activity and IgG or remission.
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The Lyme disease spirochete, Borrelia burgdorferi, can be recovered long after initial infection, even from antibiotic-treated patients, indicating that it resists eradication by host defense mechanisms and antibiotics. Since B. burgdorferi first infects skin, the possible protective effect of skin fibroblasts from an antibiotic commonly used to treat Lyme disease, ceftriaxone, was examined. Human foreskin fibroblasts protected B. burgdorferi from the lethal action of a 2-day exposure to ceftriaxone at 1 µg/mL, 10–20 × MBC. In the absence of fibroblasts, organisms did not survive. Spirochetes were not protected from ceftriaxone by glutaraldehyde-fixed fibroblasts or fibroblast lysate, suggesting that a living cell was required. The ability of the organism to survive in the presence of fibroblasts was not related to its infectivity. Fibroblasts protected B. burgdorferi for at least 14 days of exposure to ceftriaxone. Mouse keratinocytes, HEp-2 cells, and Vero cells but not Caco-2 cells showed the same protective effect. Thus, several eukaryotic cell types provide the Lyme disease spirochete with a protective environment contributing to its long-term survival.
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This article has no abstract; the first 100 words appear below. THE survival of intracellular pathogens in the host depends on their ability to avoid being killed before or after they invade mononuclear phagocytes; these cells are critical in this respect. In some circumstances the organisms are able to replicate intracellularly; this results in their dissemination and in disease.¹ The phenomenon of facultative intracellular parasitism is found in many classes of infectious agents, including viruses (herpesvirus, cytomegalovirus, and the human immunodeficiency virus), bacteria (listeria, legionella, and mycobacteria), fungi (histoplasma and cryptococcus), and protozoa (toxoplasma and leishmania). Despite the marked biologic differences among these organisms, they use a common pathway to establish . . . Adel A.F. Mahmoud Case Western Reserve University, Cleveland, OH 44106
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The later stages of infection by the Lyme disease pathogen, Borrelia burgdorferi, are characterized by the persistence of the organism in individuals possessing a strong anti-Borrelia immune response. This suggests that the organism is sequestered in a tissue protected from the immune system of the host or there is a reservoir of the organism residing within the cells of the host. In this report, the ability of B. burgdorferi to gain entrance into human umbilical vein endothelial cells was explored as a model for invasion. Incubation of B. burgdorferi with human umbilical vein endothelial cells at ratios ranging from 200:1 to 5,000:1 resulted in the intracellular localization of 10 to 25% of B. burgdorferi in 24 h. The intracellular location of the spirochetes was demonstrated by the incorporation of radiolabeled B. burgdorferi into a trypsin-resistant compartment and was confirmed by double-immunofluorescence staining which differentiated intracellular from extracellular organisms. Actin-containing microfilaments were required for the intracellular localization, indicating that the host cell participates in the internalization process. Activation of endothelial cells by agents known to increase the expression of several adhesion molecules had no effect on the interaction of B. burgdorferi with the endothelial monolayer. This indicates that the endothelial receptor for B. burgdorferi is constitutively expressed and that internalization is not dependent upon adhesion molecules whose expression is induced by inflammatory mediators. The demonstration of B. burgdorferi within endothelial cells suggest that intracellular localization may be a potential mechanism by which the organism escapes from the immune response of the host and may contribute to persistence of the organism during the later stages of Lyme disease.
Article
The chronic inflammatory condition that develops after infection by B. burgdorferi is a complex process resulting from host responses to a limited number of organisms. Amplification mechanisms driven by potent proinflammatory molecules, i.e., IL-1, may explain the vigorous response to a paucity of organisms. Spirochete dissemination to distant locations involves adherence to and penetration across endothelium and may be facilitated by host responses that increase vessel permeability. The apparent lack of tissue tropism in Lyme disease is reflected in the organism's ability to adhere to different eucaryotic cell types in vitro and the wide distribution of B. burgdorferi in various organs of infected humans and experimentally infected animals. While phagocytosis and complement activation have been observed in vitro, the specific immune response that develops in humans is inefficient in eradicating the organisms, which may possess some mechanism(s) to evade this response. There is significant evidence for host autoreactivity based on antigenic cross-reactivity between the 41-kDa flagellar subunit and stress proteins of the spirochetes and endogenous host cell components. Although the outer surface proteins appear to be suitable candidates as targets for vaccination in animal studies, fundamental differences in the immune response to spirochetal components may preclude their use in humans.
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The natural history of Lyme disease is not completely known. We studied the long-term course of Lyme arthritis in 46 children in whom the onset of the disease occurred between 1976 and 1979 and who received no antibiotic therapy for at least the first four years of the illness. Of the 46 children (age range, 2 to 15 years), 33 (72 percent) initially had erythema migrans, 7 (15 percent) had influenza-like symptoms, and 6 (13 percent) had migratory joint pain. These manifestations were followed by brief attacks of arthritis, particularly affecting the knee. The percentage of children with recurrent episodes of arthritis declined each year. By year 4, only 10 children still had a mean of two episodes of arthritis per year; the duration of arthritis was generally longer in older children (P less than 0.05). During the sixth year of illness, two children (4 percent) had keratitis, and more than 10 years after the onset of disease, a subtle encephalopathy developed in two other children. Of the 39 children whom we were able to contact in 1988-1989, 12 (31 percent) still had occasional brief episodes of joint pain and 1 (3 percent) had marked fatigue. All 46 children had positive IgG antibody responses to Borrelia burgdorferi throughout the illness and on long-term follow-up. As compared with those who became asymptomatic, the children with recurrent symptoms more often had IgM responses to the spirochete and had significantly higher IgG titers (P less than 0.05). The course of initially untreated Lyme disease in children may include acute infection followed by attacks of arthritis and then by keratitis, subtle joint pain, or chronic encephalopathy.
Article
We describe a woman who suffered for several years from joint pain with subsequent development of arthritis of her left knee. Because of these symptoms she was subjected to repeated studies including arthroscopy and menisectomy. IgG antibodies to Borrelia determined by ELISA were reported to be present and showed an increase of IgG titer to 1:1024. Histology from the last synovial biopsy disclosed only evidence of a nonspecific synovitis and marked inflammatory infiltration with lymphocytes and plasma cells. Borrelia-like structures were detected electron microscopically on semithin Epon sections from the lumen of the strongly infiltrated blood capillary. In our case the detection of Borrelia indicated the basis for increase of antibodies against Borreliae. It established the correct diagnosis and led to specific therapy of the disease after several years of unsuccessful attempts to control it.
Article
Cardiac involvement occurring early in Borrelia burgdorferi infection is a clinical manifestation of human Lyme disease. Therefore, two patients with acute complete atrioventricular heart blocks and unexplained recurrent dizziness were studied. Both patients had significantly elevated serum titers of IgM and IgG antibodies to B. burgdorferi. Right ventricular subendocardial biopsies showed dense infiltrates consisting of lymphocytes and plasma cells. Silver staining revealed spirochetes characteristic of B. burgdorferi near and in the infiltrates, between the muscle fibers, and in the endocardium. One patient responded to penicillin; the other did not, necessitating installation of a pacemaker. Thus, permanent heart damage may result from cardiac involvement in Lyme disease.
Article
Erythema migrans (EM), the distinctive cutaneous lesion of Lymedisease, has a variable clinical appearance, but at some point presents as a centrifugally expanding, usually erythematous, annular patch. Of 237 patients with this condition, 201 (85%) were examined initially from May through September. Thirty-four (14%) remembered having been bitten by a deer tick. The median interval from the bite to the appearance of EM was 9 days (range, 1–36 days). Forty-one (17%) of the patients had multiple EM lesions. Of the 237 patients, 128 (54%) manifested major extracutaneous signs and symptoms. Although EM also has a variable histologic picture, the presence of a deep and superficial perivascular and interstitiallymphohistiocytic infiltrate containing plasma cells is diagnostic. Spirochetes can be demonstrated with Warthin-Starry staining in ∼40% of the biopsy specimens. Concomitant cutaneous lesions appeared on some patients before and during antibiotic therapy. Nine patients with serologic evidence of Borrelia burgdorferi infection had cutaneous lesions other than EM, including granuloma annulare (three), erythema nodosum (two), papular urticaria (two), Henoch-Schonlein-like purpura (one), and morphea (one). Whether these entities are cutaneous markers of Lyme disease or are coincidental findings is yet to be determined.
Article
Although indirect evidence suggests that chronic Lyme arthritis is caused by persistent infection with Borrelia burgdorferi, direct visualization has been lacking. We report the demonstration of B. burgdorferi from synovial fluid aspirated from the right knee of a 31-year-old man with Lyme arthritis for more than 1 year. After 6 days, culture medium inoculated with synovial fluid showed one motile and several nonmotile spirochetes. Direct immunofluorescence staining showed reactivity with anti-B. burgdorferi serum. Spirochetes were not seen in subcultured material. The patient's arthritis improved with high-dose intravenous penicillin. Identification of B. burgdorferi from the joint fluid of a patient with long-standing arthritis supports the concept that the arthritis is due to persistent infection.
Article
To determine the clinical evolution of Lyme arthritis, 55 patients who did not receive antibiotic therapy for erythema chronicum migrans were followed longitudinally for a mean duration of 6 years. Of the 55 patients, 11 (20%) had no subsequent manifestations of Lyme disease. From 1 day to 8 weeks after disease onset, 10 of the patients (18%) began to have brief episodes of joint, periarticular, or musculoskeletal pain for as long as 6 years, but they never developed objective joint abnormalities. From 4 days to 2 years after disease onset, 28 (51%) had one episode or began to have intermittent attacks of frank arthritis, primarily in large joints; a few had polyarticular movement. The total number of these patients who continued to have recurrences decreased by 10% to 20% each year. The remaining 6 patients (11%) developed chronic synovitis later in the illness; of these, 2 (4%) had erosions, and 1 (2%), permanent joint disability. The spectrum of Lyme arthritis ranges from subjective joint pain, to intermittent attacks of arthritis, to chronic erosive disease.
Article
1. Certain aspects of the Jarisch—Herxheimer reaction (JHR) after treatment of louse-borne relapsing fever have been studied in nine patients. Peripheral vasoconstriction immediately preceded the chill phase and profound vasodilation accompanied the flush phase. 2. From four patients 50–150 ml of blood was taken at the onset of the chill and reinjected intravenously into the same patient the next day. One patient experienced a reaction identical with JHR 40–60 min after reinjection, suggesting that the mediator of JHR was present in that blood. 3. From five patients 40 ml of blood were taken serially throughout JHR and 2-ml samples of plasma, free of cells and spirochaetes, were injected into pairs of normal rabbits. All samples taken while the patients had fever were pyrogenic. After incubation with normal rabbit plasma these samples failed to produce fever in endotoxin-refractory rabbits. 4. These results suggest that in the plasma of patients with relapsing fever there is a powerful endotoxin. We were unable to show that the concentration of endotoxin increased during JHR, nor could we demonstrate the presence of leucocyte pyrogen. This failure could be a problem of quantity of plasma used or of an endotoxin-refractory state. 5. The bearing of these observations on the mechanism of JHR and the uselessness of cortisol in modifying the reaction are discussed.
Article
Abundant evidence suggests that Treponema pallidum (T.p.) escapes humoral immune defence despite the host produces antibodies early in the infection. Since the serologic responses in syphilis have been studied in detail this paper focuses on the cellular immune mechanisms. For this purpose the leukocyte migration inhibition was investigated in 17 patients in different stages of syphilis. Leukocyte migration inhibition assay was performed before, and 7 days, 3 weeks, 2 mo, and 1 yr after start of treatment. Ultrasonicated T.p. were used as antigen corresponding to 5 × 10⁶ to 2 × 10⁷ Treponema pallida per ml. Controls without antigen, with addition of Concanavalin A instead of T.p. and using cells of normal volunteers were run. There was no leukocyte migration inhibition before treatment, suggesting nonexistent or depressed cellular immunity in the untreated syphilitic patient. Significant leukocyte migration inhibition, however, was observed as early as 2 days after start of treatment, being most pronounced after 1 week. Hypothetical circulating blocking factors for cellular immune reactions might be present in the untreated syphilitic patient, which become abolished after therapy. Since stimulation with Con A of syphilitic leukocytes gave normal results even before treatment in the syphilitic patient, there might be a specific block of leukocyte migration inhibition against T.p.
Article
Lyme disease (LD) is a tick-borne spirochetal infection with a wide range of neurologic and non-neurologic manifestations. The clinical diversity of LD and limitations in serologic diagnosis often make it difficult to document the diagnosis of neuroborreliosis with certainty. We reviewed clinical manifestations in 97 seropositive children with particular attention to neurologic manifestations. Diagnostic criteria used in other case surveys were applied to determine how often a definitive diagnosis of neuroborreliosis could be made in children. Of 69 children who met criteria for LD, 32% (22) had new neurologic signs, 73% (16) of which were accounted for by facial palsy and aseptic meningitis. Five of those with neurologic findings also had erythema migrans (EM), and one had both EM and arthritis. Among those with neurologic involvement, boys outnumbered girls two to one. Neurologic abnormalities resolved spontaneously in five children before their serologic results were known. In our series, only 27% of children with neurologic abnormalities due to LD had a history of EM or arthritis. Seropositivity commonly constituted the primary basis for diagnosis of LD. Despite its nonspecificity, seropositivity for LD in children with neurologic symptoms usually signifies active neuroborreliosis.
Article
Antibiotic therapy with penicillin, doxycycline, and ceftriaxone has proven to be effective for the treatment of Lyme borreliosis. In some patients, however, it was noticed that borreliae can survival in the tissues in spite of seemingly adequate therapy. For a better understanding of this phenomenon, we investigated the different modes of degeneration of Borrelia burgdorferi suspensions during a 96-h exposure to various antibiotics. By dark-field microscopy and ultrastructural investigations, increasing blebbing and the gradual formation of granular and cystic structures could be followed during the exposure time. Although antibiotic concentrations at the MIC at which 90% of organisms are inhibited after 72 h were 80% or even greater, motile organisms were still present after incubation with penicillin and doxycycline but not after incubation with ceftriaxone. By transmission electron microscopy, intact spirochetal parts, mostly situated in cysts, were seen up to 96 h after exposure with all three antibiotics tested. According to experiences from studies with other spirochetes it is suggested that encysted borreliae, granules, and the remaining blebs might be responsible for the ongoing antigenic stimulus leading to complaints of chronic Lyme borreliosis.
Article
We monitored the antibody responses of 55 treated patients with early Lyme disease and physician-documented erythema migrans. Six sequential serum samples were obtained from patients before, during, and until one year after antibiotic therapy and analyzed by in-house enzyme-linked immunosorbent (ELISA) and immunoblot assays. An immunoblot procedure utilizing a gradient gel and an image analysis system was developed. A relational database management system was used to analyze the results and provide criteria for early disease immunoblot interpretation. Recommended criteria for the immunoglobulin M (IgM) immunoblot are the recognition of two of three proteins (24, 39, and 41 kDa). The recommended criteria for a positive IgG immunoblot are the recognition of two of five proteins (20, 24 [> 19 intensity units], 35, 39, and 88 kDa). Alternatively, if band intensity cannot be measured, the 22-kDa protein can be substituted for the 24-kDa protein with only a small decrease in sensitivity. Monoclonal antibodies were used to identify all these proteins except the 35-kDa protein. With the proposed immunoblot interpretations, the sequential serum samples were examined. At visit 1, the day of diagnosis and initiation of treatment, 54.5% of the serum samples were either IgM or IgG positive. The peak antibody response, with 80% of the serum samples positive, occurred at visit 2, 8 to 12 days into treatment. The sensitivities of the IgM and IgG immunoblot for detecting patients that were seropositive into the study period were 58.5 and 54.6%, respectively, at visit 1 and 100% at visit 2. Twenty percent of the patients remained seronegative throughout the study. The specificities of the IgM and IgG immunoblots were 92 to 94% and 93 to 96%, respectively. The IgM immunoblot and ELISA were similar in sensitivities, whereas the IgG immunoblot had greater sensitivity than the IgG ELISA (P = 0.006).
Article
Three patients with leptospirosis whose condition worsened after initiation of antibiotic therapy are reported. Their clinical deterioration appeared to be due to the development of the Jarisch-Herxheimer reaction rather than to progression of their underlying infection. Relevant aspects of the management of patients with leptospirosis are discussed.
Article
There are currently no accepted criteria for positive Western blots in Lyme disease. In a retrospective analysis of 225 case and control subjects, the best discriminatory ability of test criteria was obtained byrequiring at least 2 of the 8 most common IgM bands in early disease (18, 21,28,37,41,45,58, and 93 kDa) and by requiring at least 5 ofthe 10 most frequent IgG bands afterthe first weeks of infection (18, 21, 28, 30, 39, 41, 45, 58, 66, and 93 kDa). When these definitions weretested in a prospective study of all 237 patients seen in a diagnostic Lyme disease clinic during a 1-year period and in 74 patients with erythema migrans or summer flu-like illnesses, the IgM blot in early disease had a sensitivity of 32% and a specificity of 100%; the IgG blot after the first weeks of infection had a sensitivity of 83% and a specificity of 95%. Among patients with indeterminate IgG responses by ELISA, 6 of 9 patients with active Lyme disease had positive blots compared with 2 of 34 patients with other illnesses (P < .001). Thus, Western blotting can be used to increase the specificity of serologic testing in Lyme disease.
Article
The ability of Borrelia burgdorferi to attach to and invade human fibroblasts was investigated by scanning electron and confocal microscopy. By scanning electron microscopy, B. burgdorferi were tightly adherent to fibroblast monolayers after 24-48 h but were eliminated from the cell surface by treatment with ceftriaxone (1 µg/mL) for 5 days. Despite the absence of visible spirochetes on the cell surface after antibiotic treatment, viable B. burgdorferi were isolated from lysates of the fibroblast monolayers. B. burgdorferi were observed in the perinuclear region within human fibroblasts by laser scanning confocal microscopy. Intracellular spirochetes specifically labeled with monoclonal anti-flagellin antibody were also identified by fluorescent laser scanning confocal microscopy. These observations suggest that B. burgdorferi can adhere to, penetrate, and invade human fibroblasts in organisms that remain viable.
Article
In patients with louse-borne relapsing fever (Borrelia recurrentis infection), antimicrobial treatment is often followed by sudden fever, rigors, and persistent hypotension (Jarisch-Herxheimer reactions) that are associated with increases in plasma concentrations of tumor necrosis factor alpha (TNF-alpha), interleukin-6, and interleukin-8. We attempted to determine whether sheep polyclonal Fab antibody fragments against TNF-alpha (anti-TNF-alpha Fab) could suppress the Jarisch-Herxheimer reaction. We conducted a randomized, double-blind, placebo-controlled trial in 49 patients with proven louse-borne relapsing fever. Immediately before the intramuscular injection of penicillin, the patients received an intravenous infusion of either anti-TNF-alpha Fab or a control solution. Ten of the 20 patients given anti-TNF-alpha Fab had Jarisch-Herxheimer reactions with rigors, as compared with 26 of the 29 control patients (P = 0.006). The controls had significantly greater mean maximal increases in temperature (1.5 vs. 0.8 degrees C, P < 0.001), pulse rate (31 vs. 13 per minute, P < 0.001), and systolic blood pressure (25 vs. 15 mm Hg, P < 0.003), as well as higher mean peak plasma concentrations of interleukin-6 (50 vs. 17 micrograms per liter) and interleukin-8 (2000 vs 205 ng per liter) (P < 0.001 for both comparisons). Levels of TNF-alpha were undetectable after treatment with anti-TNF-alpha Fab. Pretreatment with sheep anti-TNF-alpha Fab suppresses Jarisch-Herxheimer reactions that occur after penicillin treatment for louse-borne relapsing fever, reduces the associated increases in plasma concentrations of interleukin-6 and interleukin-8, and may be useful in other forms of sepsis.
Article
To investigate if Borrelia burgdorferi can persist in resident joint cells, an infection model using cell cultures of human synovial cells was established and compared to the interaction of Borrelia burgdorferi and human macrophages. Borrelia burgdorferi were found attached to the cell surface or folded into the cell membrane of synovial cells analysed by transmission electron and confocal laser scanning microscopy. In contrast to macrophages, morphologically intact Borrelia burgdorferi were found in the cytosol of synovial cells without engulfment by cell membrane folds or phagosomes. Borrelia burgdorferi were isolated from parallel cultures. Treatment with ceftriaxone eradicated extracellular Borrelia burgdorferi, but spirochetes were reisolated after lysis of the synovial cells. Borrelia burgdorferi persisted inside synovial cells for at least 8 weeks. These data suggested that Borrelia burgdorferi might be able to persist within resident joint cells in vivo.
Article
To perform the first systematic electronmicroscopic (EM) and immunoelectron microscopy (IEM) study of the pathological changes and the evidence of spirochete presence in synovial membranes and synovial fluid (SF) cells of patients with chronic Lyme arthritis. EM examination was performed on four synovial membrane and eight SF cell samples from eight patients with chronic Lyme disease. Spirochetal antigens in the samples were sought by IEM using monoclonal antibody to Borrelia burgdorferi outer surface protein A (OspA) as the immunoprobe. Prominent ultrastructural findings were surface fibrin-like material, thickened synovial lining cell layer and signs of vascular injury. Borrelia-like structures were identified in all four synovial membranes and in two of eight SF cell samples. The presence of spirochetal antigens was confirmed by IEM in all four samples studied (one synovial membrane and three SF cell samples). OspA labelling was in perivascular areas, deep synovial stroma among collagen bundles, and in vacuoles of fibroblasts in synovial membranes; and in cytophagosomes of mononuclear cells in SF cell samples. Electron microscopy adds further evidence for persistence of spirochetal antigens in the joint in chronic Lyme disease. Locations of spirochetes or spirochetal antigens both intracellulary and extracellulary in deep synovial connective tissue as reported here suggest sites at which spirochaetes may elude host immune response and antibiotic treatment.
Article
The reliability of various in vitro techniques to identify Borrelia burgdorferi infection is still unsatisfactory. Using a high-power resolution videomicroscope and staining with the borrelia genus-specific monoclonal flagellar antibody H9724, we identified borrelial structures in skin biopsies of erythema chronicum migrans (from which borrelia later was cultured), of acrodermatitis chronica atrophicans, and of morphea. In addition to typical borreliae, we noted stained structures of varying shapes identical to borreliae found in a "borrelia-injected skin" model; identical to agar-embedded borreliae; and identical to cultured borreliae following exposure to hyperimmune sera and/or antibiotics. We conclude that the H9724-reactive structures represent various forms of B. burgdorferi rather than staining artifacts. These "atypical" forms of B. burgdorferi may represent in vivo morphologic variants of this bacterium.
Article
In 1991, we reported that 55% of laboratories participating in the Wisconsin Proficiency Testing Program could not accurately identify serum samples from Lyme disease patients containing antibody against Borrelia burgdorferi. The purpose of this study was to determine whether the accuracy of Lyme disease test results reported by approximately 500 participants in the Wisconsin State Laboratory of Hygiene/College of American Pathologists Lyme Disease Survey had improved. From 1992 through 1994, 50 serum samples were sent to participants of the survey. Each laboratory received 28 serum samples from individuals with Lyme disease according to the case definition of the Centers for Disease Control and Prevention and 22 serum samples from healthy individuals. Unfortunately, the serodiagnosis of Lyme disease by participants had not improved. The specificity of the Lyme disease assays steadily decreased from approximately 95% to approximately 81% during the 3-year period of the survey. False-positive test results approached 55% with some of the serum samples from healthy donors. A serum sample containing antibody against Treponema pallidum was reported as positive by 70% of the participants. In addition, the sensitivity fluctuated between 93 and 75%, depending upon the conjugate used by the laboratories. These results suggest that stronger criteria must be applied for approving and continuing to approve commercially available kits for the serodiagnosis of Lyme disease.
Article
Two hundred seventy-seven patients with chronic Lyme disease were treated with tetracycline for 1 to 11 months (mean, 4 months); the outcomes for these patients were generally good. Overall, 20% of the patients were cured; 70% of the patients' conditions improved, and treatment failed for 10% of the patients. Improvement frequently did not take place for several weeks; after 2 months of treatment, 33% of the patients' conditions were significantly improved (degree of improvement, 75%–100%), and after 3 months of treatment, 61% of the patients' conditions were significantly improved. Treatment outcomes for seronegative patients (20% of all patients) were similar to those for seropositive patients. Western immunoblotting showed reactions to one or more Borrelia burgdorferi-specific proteins for 65% of the patients for whom enzyme-linked immunosorbent assays were negative. Whereas age, sex, and prior erythema migrans were not correlated with better or worse treatment outcomes, a history of longer duration of symptoms or antibiotic treatment was associated with longer treatment times to achieve improvement and cure. These results support the use of longer courses of treatment in the management of patients with chronic Lyme disease. Controlled trials need to be conducted to validate these observations.
Article
Lyme disease is a persistent low-density spirochetosis caused by Borrelia burgdorferi sensu lato. Although spirochetes causing Lyme disease are highly immunogenic in experimental models, the onset of specific antibody responses to infection is often delayed or undetectable in some patients. The properties and mechanisms mediating such immune avoidance remain obscure. To examine the nature and consequences of interactions between Lyme disease spirochetes and immune effector cells, we coincubated B. burgdorferi with primary and cultured human leukocytes. We found that B. burgdorferi actively attaches to, invades, and kills human B and T lymphocytes. Significant killing began within 1 hour of mixing. Cytopathic effects varied with respect to host cell lineage and the species, viability, and degree of attenuation of the spirochetes. Both spirochetal virulence and lymphocytic susceptibility could be phenotypically selected, thus indicating that both bacterial and host cell factors contribute to such interactions. These results suggest that invasion and lysis of lymphocytes may constitute previously unrecognized factors in Lyme disease and bacterial pathogenesis.
Article
The purpose of this study was to evaluate the behaviour of Borrelia burgdorferi under controlled conditions. The occurrence of cystic forms of Borrelia burgdorferi in vitro was noted, and these cysts were able to be transformed to normal, mobile spirochetes. B. burgdorferi was cultivated in a commercial culture medium without serum. The spirochetes multiplied only slowly in this medium, and transformation to encysted forms was observed after 1 week. When these cysts were transferred to the same culture medium with rabbit serum, the encysted forms developed into regular, mobile spirochetes after 6 weeks, and their regeneration time was normal. Examination of these cysts in the transmission electron microscope revealed transverse fission inside the cysts. It is probable that similar phenomena may occur in vivo under conditions unfavourable for spirochetes. These observations may help to explain why diagnosis and treatment of B. burgdorferi infections in humans can be difficult.
Article
Heat-killed Borrelia burgdorferi spirochetes stimulate in vitro production of interleukin-10 (IL-10) at both mRNA and protein levels in peripheral blood mononuclear cells (PBMC) of uninfected rhesus monkeys. A concomitant down-modulation of IL-2 gene transcription was observed. Neither IL-4 nor gamma interferon gene expression was ostensibly affected by B. burgdorferi spirochetes. These phenomena were observed regardless of whether the stimulating spirochetes belonged to the B. burgdorferi sensu stricto, Borrelia afzelii, or Borrelia garinii genospecies, the three main species that cause Lyme disease. B. burgdorferi also induced production of IL-10 in uninfected human PBMC, indicating that this effect might play a role in human Lyme disease. Purified lipidated outer surface protein A (OspA), but not its unlipidated form, induced the production of high levels of IL-10 in uninfected human PBMC. Thus, the lipid moiety is essential in the induction of IL-10 in these PBMC. B. burgdorferi M297, a mutant strain that lacks the plasmid that encodes OspA and OspB, also induced IL-10 gene transcription in PBMC, indicating that this phenomenon is not causally linked exclusively to OspA and its lipid moiety. These results demonstrate that B. burgdorferi can stimulate the production of an antiinflammatory, immunosuppressive cytokine in naive cells and suggest that IL-10 may play a role both in avoidance by the spirochete of deleterious immune responses and in limiting the inflammation that the spirochete is able to induce.
Article
A 31-year-old woman diagnosed with Lyme disease was treated with amoxicillin. One hour after the first antibiotic dose, the patient became acutely ill. She developed hypertension, fever, and rigors. Shortly afterward, she became hypotensive and required fluid resuscitation. This systemic illness, the Jarisch-Herxheimer reaction, was first noted in association with antibiotic therapy for neurosyphilis. Thus, the institution of antibiotic therapy may be complicated by the Jarisch-Herxheimer reaction.
Article
The purpose of this study was to examine the structural alterations of Borrelia burgdorferi when exposed to spinal fluid. Normal, mobile spirochetes were inoculated into spinal fluid, and the spirochetes were converted to cysts (spheroplast L-forms) after 1-24 h. When these cystic forms were transferred to a rich BSK-H medium, the cysts were converted back to normal, mobile spirochetes after incubation for 9 to 17 days. The cultures were examined by dark field microscopy (DFM), interference contrast microscopy (ICM) and transmission electron microscopy (TEM). When neuroborreliosis is suspected, it is necessary to realize that B. burgdorferi can be present in a cystic form, and these cysts have to be recognized by microscopy. This study may also explain why cultivation of spinal fluid often is negative with respect to B. burgdorferi.
Article
A total of 165 patients with disseminated Lyme borreliosis (diagnosed in 1990-94, all seropositive except one culture-positive patient) were followed after antibiotic treatment, and 32 of them were regarded as having a clinically defined treatment failure. Of the 165 patients, 136 were tested by polymerase chain reaction (PCR) during the follow-up. PCR was positive from the plasma of 14 patients 0-30 months after discontinuation of the treatment, and 12 of these patients had a clinical relapse. In addition, Borrelia burgdorferi was cultured from the blood of three patients during the follow-up. All three patients belonged to the group with relapse, and two of them were also PCR positive. This report focuses on the 13 patients with clinical relapse and culture or PCR positivity. Eight of the patients had culture or PCR-proven initial diagnosis, the diagnosis of the remaining five patients was based on positive serology only. All 13 patients were primarily treated for more than 3 months with intravenous and/or oral antibiotics (11 of them received intravenous ceftriaxone, nine for 2 weeks, one for 3 weeks and one for 7 weeks, followed by oral antibiotics). The treatment caused only temporary relief in the symptoms of the patients. All but one of them had negative PCR results immediately after the first treatment. The patients were retreated usually with intravenous ceftriaxone for 4-6 weeks. None of them was PCR positive after the retreatment. The response to retreatment was considered good in nine patients. We conclude that the treatment of Lyme borreliosis with appropriate antibiotics for even more than 3 months may not always eradicate the spirochete. By using PCR, it is possible to avoid unnecessary retreatment of patients with 'post-Lyme syndrome' and those with 'serological scars' remaining detectable for months or years after infection.