Volume: 1 Table of Contents: Laboratory Diagnostic Tests for Lyme Disease by David W. Dorward, Ph. D. [email protected] Senior Staff Fellow Laboratory of Vectors and Pathogens NIH/Rocky Mountain Laboratories Hamilton, MT 59840 Newsletter: ***************************************************************************** * Lyme Disease Electronic Mail Network * * LymeNet Newsletter * ***************************************************************************** Volume 1 - Number 05 - 3/10/93 ***** SPECIAL ISSUE ***** I. Introduction II. News from the wires III. Jargon Index IV. How to Subscribe, Contribute and Get Back Issues I. ***** INTRODUCTION ***** This issue of the LymeNet newsletter features a paper written by Dr. David W. Dorward, Senior Staff Fellow at the NIH's Rocky Mountain Labs. This article was written for electronic publication in the Health Info-Com Network Newsletter, a biweekly newsletter edited by Dr. David Dodell. My thanks to Dr. Dodell for the reprint permission. More information on the Heath Info-Com Newsletter can be found at the end of the article. This article reviews the current state of Lyme testing. Dr. Dorward looks at the many different testing techniques, both existing and in development. The pros and cons of each testing method are discussed, as well as recent improvements. -Marc. II. ***** NEWS FROM THE WIRES ****** ----------------------------------------------------------------------------- Compilation Copyright 1993 by David Dodell, D.M.D. All rights Reserved. License is hereby granted to republish on electronic media for which no fees are charged, so long as the text of this copyright notice and license are attached intact to any and all republished portion or portions. ----------------------------------------------------------------------------- Laboratory Diagnostic Tests for Lyme Disease by David W. Dorward, Ph. D. [email protected] Senior Staff Fellow Laboratory of Vectors and Pathogens NIH/Rocky Mountain Laboratories Hamilton, MT 59840 _______________________________ INTRODUCTION Lyme disease is an emerging disease, which has become the most prevalent arthropod-borne disease in North America. It is a protean disease, and can cause a mild to severe, acute to chronic syndrome in humans and in wild and domestic animals. Despite considerable efforts to understand, recognize, diagnose, and treat Lyme disease, many questions remain, particularly regarding methods used for laboratory confirmation of infection. In most cases, the results of laboratory testing methods are consistent with clinical presentation of Lyme disease. However, when inconsistent, little confidence is placed on the laboratory results because of the many published reports of relatively high degrees of error among test systems. In recent years several new testing procedures have been proposed to address such problems. This paper describes the theoretical bases for current and "next generation" Lyme test systems and discusses the benefits, potential problems, and recent improvements with each type of system. The emphasis of the descriptions and discussions of current assays focuses on systems which have been developed and marketed by manufacturers listed and which have been cleared for clinical use in the United States by the U.S. Food and Drug Administration (FDA). Readers should note that many additional systems can be and are utilized by individual clinical laboratories. Furthermore, several additional companies supply test systems which have previously received clearance by the FDA, after being submitted for consideration by manufacturers listed herein. Review of these systems follows a brief general description of Lyme disease. In just over a decade since the discovery of a spirochetal etiology for Lyme disease, scientific interest in Lyme disease has led to a dramatic increase in knowledge about the disease and it's agents, three members of Borrelia sp. Originally isolated and identified by Willy Burgdorfer and co-workers (1), these agents were initially named Borrelia burgdorferi. More recently, evidence of phylogenetic diversity among Lyme disease associated spirochetes from the United States, Europe, and Asia, has led to acceptance of a proposal for subdividing the agents into separate species, designated B. burgdorferi, B. garinii, and "group VS461" (2). Lyme spirochetes are transmitted to humans and other mammals by several species of hard-shell ticks primarily those within the Ixodes ricinus complex, comprising I. ricinus and I. persulcatus in Eurasia, and I. dammini, I. scapularis, I. pacificus, and possibly I. cookei in North America. There is also evidence to suggest that Amblyomma americanum may also be a competent vector in the southern United States. The spirochetes gain entry to mammals during feeding by these ticks. Most human infections occur after bites by the exceedingly tiny larval and nymphal stages of Ixodes sp. ticks, which emerge in late spring to early summer. Hence, Lyme disease is most prevalent in areas inhabited by these ticks, and occurs most frequently from May to September. Difficulties in diagnosis arise from several factors. Such factors include variability in the rate of onset and the types of symptoms exhibited, failure of patients to see or remember tick bites, slow growth and low density of infecting spirochetes, and differences in host responses to the infection. Comprehensive reviews of these factors have previously been published (3, 4). The U.S. Centers for Disease Control (CDC) has also released "case definition" guidelines for Lyme disease. Typically (in 50-80% of cases), within several days of a tick bite, a characteristic and pathognomonic "bulls-eye" rash, called erythema migrans, will encircle the site of the bite. Clinical isolation of spirochetes is often possible from skin biopsies taken at or near the margins of erythema migrans lesions. In cases in which erythema migrans did not appear or was not noticed, isolation of spirochetes is relatively rare (usually less than 10% of cases). In those situations diagnoses are based upon clinical observations, patient history, presence of secondary symptoms such as arthritis, arthralgias, carditis, and neurological manifestations, and results of clinical laboratory tests. With few exceptions, the laboratory tests involve determinations of diagnostic titers of host antibodies directed against the Lyme agents, either by indirect fluorescent assay (IFA), enzyme-linked immunoassay (EIA), or Western blot analysis (WBA). For reporting, the CDC case definition requires one or more of the following indications: a) isolation of the causative spirochetes, b) presence of erythema migrans, or c) a combination of "late" symptoms and laboratory confirmation by serology as described above. ___________________________________ LABORATORY DIAGNOSTIC STRATEGIES Laboratory assays for the diagnosis of Lyme disease, which are in practice or which have been proposed, can be grouped into the following categories: 1) Isolation and culture of agents 2) Histological examination of tissues 3) Serology 4) Antigen detection systems 5) Nucleic acid detection systems Most of the assays in the categories 3-5 are considered "rapid" tests, and can be performed under normal circumstances in less than one day. Such tests are the focus of most research into Lyme diagnostics. Furthermore, all commercial test systems currently cleared by the FDA for clinical use in the U.S. fall within the serological category. Hence this discussion of rapid test systems will concentrate on categories 3-5, with a brief description of categories 1-2. 1) Isolation Attempts to isolate Lyme agents from patients are common, and certainly conclusive when successful. The ability to culture the spirochetes from human patients is highly variable. Furthermore, primary cultures generally require 1-2 weeks or more before growth of the spirochetes is evident. The source of the sample factors into whether the spirochetes can be cultured. Successful cultures have been obtained from ticks, skin biopsies, ear punches, cerebrospinal fluid, blood, and synovial fluid (3, 4). In animal models, the spirochetes are reliably cultured from ticks, and from heart muscle and the urinary bladder of mice (3, 4). To help avoid contamination of cultures, samples are handled carefully and antibiotics are usually added to primary culture media. 2) Histology Histological examination of patient tissues and blood smears has been and continues to be used in some laboratories as a diagnostic assay. Histological examinations are usually coupled with immunological assays such as fluorescence microscopy or immune histochemistry to enhance the specificity of the assay. Since Lyme disease is characterized by a low density infection, examination of large numbers of sections is often required before spirochetes are observed. Similarly, unlike relapsing fever borreliae, Lyme spirochetes are rarely observed in blood smears. Thus, although histological observation of Lyme spirochetes can successfully confirm an infection, failure to observe the spirochetes does not necessarily rule out Lyme disease. 3) Serology Serological assays for Lyme diagnosis include IFA, EIA, WBA, and spirochetal neutralization assays. Currently all commercial test systems which have received clearance by the FDA are either IFA or EIA (Tables 1 and 2). Tables 1 and 2 compare the formats, specificities, and scope of the assays which are currently marketed and cleared. The kits have been arranged alphabetically by manufacturer. As noted above, the tables do not list products which have received clearance through one manufacturer but which are produced, repackaged, or supplied under a different label. The information shown was provided by the manufacturers. Among the fluorescent systems shown in table 1, most assays involve probing fixed spirochetes with patient sera, labeling any immune complexes formed with a fluorescent conjugate, and examining the preparation by fluorescent microscopy. Fluorescent "dot" and microtiter well assays utilizing whole cell lysates have also been developed. Approximately equal numbers of the assays detect either IgG or IgM class antibodies in the sera, and most detect both classes. Table 2 shows that most enzyme-linked systems are also similar to each other. Most have a microtiter well format to which whole cell antigens are immobilized. Dot assays and an automated system are also available. Some manufacturers have supplemented (or replaced) the whole cell antigens with spirochetal membrane concentrates, the 41 kilodalton flagellar protein, or with a recombinant 39 kilodalton antigen which is specific to Lyme spirochetes. As with fluorescent assays, approximately equal numbers of test kits detect IgG or IgM antibodies, most detect both, and some also detect IgA antibodies. Table 1. Fluorescent systems Format Antigen Detects ______________________ ____________ ___________ Mfg Slide Dot Well WC Fixed IgG IgM _________________________________________________________________ BE + + + + DT + + + + HB + + + + + + MD + + + + + + WB + + + + + + + + + + + + + + + ZS + + + + _________________________________________________________________ Abbreviations: Mfg-manufacturer; WC-Whole cell; BE-Bion Enterprises; DT-Diagnostic Technology; HB-Hillcrest Biologicals; MD- Mardx Diagnostics; WB-Whittaker Bioproducts; VS-Vitek Systems; ZS-Zeus Scientific Table 2. Enzyme-linked systems Format Antigen Detects Service ________________ ___________ _____________ Provided Mfg Well Dot Auto WC Peptide IgG IgM IgA ______________________________________________________________ CB + + + + DX + + 41k + + + + + + + 41k + + + + + + + GB + + 41k + + + 39k + + + + 39k + + + + + 39k + + + GL + + + HB + + + + + + + MD + + + + + + + + + + MT + + + + VS + + + + WB + + + + + + + ZS + + + + + + + + + + _________________________________________________________________ Abbreviations; Mfg-manufacturer; CB-Cambridge Biosciences; DX- Diamedix; GB-General Biometrics; GL-Gull Laboratories; HB-Hillcrest Biologicals; MD-Mardx Diagnostics; MT-Medical Diagnostic Technology; VS-Vitek Systems; WB-Whittaker Bioproducts; ZS-Zeus Scientific Although no commercial WBA test systems have been cleared by the FDA, many laboratories also screen sera by WBA. With WBA, laboratories can determine whether sera contain antibodies directed against a variety of different spirochetal antigens. Currently there is no clear consensus about which antigens, when recognized, are most important for making diagnostic conclusions for Lyme disease. Binding of antibodies to several proteins have been commonly observed in known Lyme sera. These include (but are not limited to) proteins at 92, 79, 60, 41, 39, 34, 31, and 22 kilodaltons (3, 4). Proteins at 31 and 34 kilodaltons, designated outer surface protein A (OspA) and OspB, respectively, are known to be plentiful in most Lyme spirochete isolates. Another, recently reported, serological approach involves testing sera for the presence of neutralizing antibodies (5). In this assay, sera are added to a test strain culture of Borrelia burgdorferi, and the ability of serum antibodies to kill the test strain is measured. 4) Antigen detections In contrast to serological systems, antigen detection systems screen for antigenic products of the spirochete rather than the host's immune response to the infection. Therefore, antigen detection systems could be expected to confirm both infections in patients with depressed immune systems and early infections in patients who have not yet produced a detectable antibody response. It is presumed that clearance of such antigens from hosts would be indicative of successful treatment and elimination of infecting spirochetes from patients. Furthermore, antigen detection systems can be utilized to monitor the presence of Lyme spirochetes in ticks. Two categories of antigen detection systems have been developed and used in laboratories. These are T- cell (T-lymphocyte) proliferative assays and antibody-based antigen detection systems. T-cell proliferative assays are based upon the recognition of spirochetal antigens by cloned, antigen-specific T-cells (6). When such T-cells bind and recognize Lyme spirochete products in a test sample, T-cell division is induced. This division can be measured by counting cells, or by measuring the incorporation of labeled nutrients into the T-cells. In theory, very few molecules of the recognized antigens would be required to induce T-cell proliferation. Monoclonal or polyclonal antibodies directed against specific Lyme spirochete antigens can also be used to detect antigens in clinical samples. Initial work found that detectable quantities of proteins including flagellin, OspA, and OspB, were excreted in the urine of infected mammals (7). Subsequent work showed that the sensitivity of detection could be enhanced by immobilizing, and effectively concentrating, the antigens onto an antibody- coated surface before detection with a species-specific antibody probe. Immobilization of antigens allows for removal of contaminants from a sample preparation which might interfere with subsequent immunological binding reactions in the assay (8). Using such an "antigen capture" system, a variety of different fluids and tissues from ticks and mammals can be screened for involvement in the infection. Mammalian fluids and tissues in which antigens have been observed include: urine, blood, cerebrospinal fluid, synovial fluid, tears, vitreous humor, breast milk, a variety of visceral organs, and the brain. Since such varied samples can contain spirochetal antigens, it is presumed that other samples could also contain detectable quantities of antigen. When using antigen capture systems, occasionally intact spirochetes are immobilized onto the antibody-activated substrate, whereas other microorganisms are excluded from adhering to the same surface (8). One approach at exploiting this capture technique used capillary tubes, coated on the inner surface with antibodies, to promote binding of spirochetes to the glass (9). Such selective adherence enabled recovery and isolation of Lyme spirochetes from biologically diverse mixtures, such as contaminated cultures, macerated ticks, and infected organs. Therefore, use of antigen capture systems may provide a means to facilitate isolating Lyme spirochetes (and potentially many other types of pathogens) from infected hosts. 5) Nucleic acid detections Like antigen detection systems, nucleic acid detection systems analyze spirochetal bioproducts, rather than a host's immune response to infection. Similarly, nucleic acid assays can also detect Lyme spirochetes in infected ticks. Currently, three types of nucleic acid detection systems have been proposed as possible diagnostic procedures for Lyme disease. These are DNA- DNA hybridizations, polymerase chain reaction (PCR), and ligase chain reaction (LCR). Hybridization assays are based upon the ability of a labeled fragment of DNA, specific to Lyme spirochetes, to locate and hybridize with complementary DNA sequences in a test sample. Chain reaction assays enable the temperature-regulated amplification and detection of spirochetal DNA molecules. Both PCR and LCR reactions incorporate the following processes a) melting of DNA strands, b) hybridization of short and species-specific single-stranded DNA primer molecules to targeted sites on templet DNA possibly present in test samples, and c) enzymatic activity by either heat stable DNA polymerase (PCR) or DNA ligase (LCR). Optimal temperatures for each step are reached and maintained by instruments called thermocyclers. Molecules amplified by PCR contain newly synthesized DNA which corresponds to sequences which lie between the binding sites for the two primer molecules. In contrast, molecules amplified by LCR result from ligation of the two primer molecules which must bind to contiguous sites on a templet. Direct DNA-DNA hybridization has been proposed and used to screen ticks for the presence of Lyme spirochetes (10). For effective detection of spirochetal DNA by hybridization, numerous target DNA molecules must usually be present in a test sample. Because few if any intact spirochetes are present in typical clinical samples, hybridization is not commonly employed to directly test for infection of mammals. Both PCR and LCR techniques for Lyme disease diagnosis are being developed (10-15). In theory, the presence of a single templet DNA molecule in a given sample would be sufficient to allow confirmation of infection by these techniques. In practice the sensitivity can usually be extrapolated to producing positive results with less than 10 templet DNA molecules in a sample. Several different studies have shown that amplification of Lyme spirochetal DNA can be accomplished from ticks, and mammalian blood, urine, and tissues. Furthermore, several target sites which are apparently specific for Lyme spirochetes have been identified (11, 12, 14, 15). These include sites within uncharacterized loci and within genes for Osp proteins, flagellin, and ribosomal RNA. Considerable interest and efforts are being exerted toward developing amplification assays for confirming infections with Lyme spirochetes, and many other microorganisms. _______________________________________ PRACTICAL CONSIDERATIONS (PROS AND CONS) 1) Isolation As with any infection, the definitive confirmatory test is isolation of the causative agent. It is also desirable to have access to isolates from patients for evaluating the antibiotic susceptibility and monitoring the epidemiology of particular isolates or strains. The low density infection and slow growth rate, characteristic of Lyme spirochetes, can present several difficulties in obtaining primary isolates. Many studies have considered methods by which to optimize the probability of obtaining spirochetal cultures from patients (3, 4). In general, the most reliable clinical samples for culturing the spirochetes are skin biopsies taken from the margins of erythema migrans lesions and from ear punches, and samples of cerebrospinal fluid. Although spirochetemia is known to occur, blood is not a reliable sample for culture. Techniques which have been developed for capturing spirochetes onto antibody-activated surfaces prior to culturing, may enable concentration of spirochetes from relatively large volume clinical samples and may help reduce the potential for contamination of cultures. 2) Histology Histological examination of clinical samples can provide conclusive evidence of infection when Lyme spirochetes are observed. Coupling the microscopic examination with an immunoassay such as immune fluorescence or immune histochemistry, can enable an evaluation of whether structures resembling spirochetes react with titrated antibodies specific for Lyme spirochetes. However, since Lyme is characterized by a low density infection, thorough histological examination of sectioned tissues can be laborious. Moreover, Lyme cannot be ruled out in patients with negative histology. No recent improvements for histological confirmation of Lyme disease have been proposed or developed. 3) Serology As noted above, all FDA-cleared and commercially available Lyme test systems are serological. In addition many public, private, and educational laboratories, have developed and use their own serological assays for Lyme disease. Serological assays are advantageous in that they are readily available; they generally produce rapid results; and guidelines for their use and interpretation are available from manufacturers, published literature, and governmental agencies such as the CDC and state health departments. Numerous studies have evaluated commercially available Lyme test kits. Among these studies there is virtually no consensus on the sensitivity and specificity, hence the accuracy and utility of the kits is brought into question. In perhaps the most comprehensive comparison of kits marketed in the U.S., the CDC evaluated the performance of 20 kits using a set of 53 human sera from patients who were clinically defined as positive or negative for Lyme disease. Figures 1 and 2 show the results of comparisons between the seven most accurate kits and the clinical definition of the samples (Fig. 1), and the degree of consistency obtained by each kit when the sera were tested at three different reference laboratories (Fig. 2). Figure 1. Sensitivity and specificity of Lyme test kits, based on clinical definition. ___________________________________________________________________ Percent: 20 40 60 80 100 ___________________________________________________________________ 1 :NNNNNNNNNNNNNN :CCCCCCCCCCCCCCCCCCCCCCCCCCCCC 2 :NNNNNNNNNNNNNNNNNNNNNNNNN :CCCCCCCCC 3 :NNNNNNNNNNNNNNNNNNNNNNNNNN :CCCCCCCCCC 4 :NNNNNNNNNNNNNNN :CCCCCCCCCCCCCCCCCCCCCCC 5 :NNNNNNNNNNNNNNNNNNNNNNNNNNNNN :CCCCCC 6 :NNNNNNNNNNNNNNNNNNN :CCCCCCCCCCCCCCCCCCCC 7 :NNNNNNNNNNNNNNNNNNNNNNNNN :CCCCCCCCCC WB :NNNNNNNNN :CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC __________________________________________________________________ Abbreviations: N-sensitivity; C-specificity; WB-western blot Source: U.S. Public Health Service, Centers for Disease Control Figure 2. Average sensitivity and specificity of Lyme test kits analyzed at three independent laboratories. __________________________________________________________________ Percent: 20 40 60 80 100 __________________________________________________________________ 1 :NNNNNNNNNNNNNNNNNNNN :CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC 2 :NNNNNNNNNNNNNNNNNNNNNNNNNNN :CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC 3 :NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN :CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC 4 :NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN :CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC 5 :NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN :CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC 6 :NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN :CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC 7 :NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN :CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC ___________________________________________________________________ Abbreviations: N-sensitivity; C-specificity; Source: U. S. Public Health Service, Centers for Disease Control Such results indicated that a significant degree of variability could be expected when these kits were used to screen patient sera for evidence of infection with Lyme spirochetes. Similar independent evaluations of test systems, performed at many different locations, have been consistent with these results. Taken together, such findings have led physicians to place limited confidence on the results of commercial test systems. Several factors could contribute to the variability and error rate associated with these systems. These include the inherent variability of patient humoral responses, the stage of infection at the time of serum sampling, the antigenic similarity of related commensal and pathogenic spirochetes (which can lead to false-positive reactions), and the antigenic variability of Lyme spirochetes within and between different geographic locations (which can result in false-negative reactions). Recently, purified spirochetal antigens or recombinant antigens have been included in some kits in an attempt to enhance the accuracy of the kits. However, evidence is mounting that a significant proportion of Lyme cases, particularly congenital cases, are seronegative. Thus, considerable effort has gone into development of alternative testing protocols. 4) Antigen detection systems Both antibody based and T-cell based antigen detection systems have been developed, but none has been cleared and marketed for clinical use. These systems offer a potential advantage over serological assays in that development of an immune response to the infection is not necessary for detection of spirochetal antigens in a sample. Such tests can also effectively screen ticks, and a variety of mammalian fluids and tissues for the presence of Lyme antigens. Like serological tests, the antibody-based antigen detection systems are also rapid. Limitations of antigen detection systems include the possibility that antigens may be excreted or remain in hosts for extended periods after effective treatment. Furthermore, these systems are vulnerable to antigenic variation among different strains and species of Lyme spirochetes, particularly when based upon recognition of individual epitopes. One recent improvement to antigenic detection systems is the use of a capture step which allows for immobilization and concentration of antigens from relatively large volumes of samples prior to testing, and can also allow for capturing intact spirochetes for subsequent aseptic isolation and culture. 5) Nucleic acid detection systems Systems for detection of spirochetal nucleic acids by hybridization, PCR, and LCR, also offer the potential advantage of being independent of the host immune response, and being applicable to screening ticks. For practical reasons discussed above, screening of samples for Lyme spirochetes by hybridization techniques are usually limited to ticks and to cultured spirochetes. Detections by PCR and LCR can be sufficiently sensitive to be used successfully on clinical samples. Furthermore, it is likely that thermocyclers will increasingly become common equipment for clinical laboratories as enzyme chain reaction diagnostic protocols are established for more pathogens. Potential problems with diagnostic use of PCR and LCR in Lyme cases include the vulnerability of the technique to mutation and genetic sequence diversity within the templet DNA target site, presence of enzyme inhibitors in clinical samples, and the potential for chance contamination of clinical samples by templet DNA molecules. Enzyme inhibitors can include a wide variety of agents such as salts, proteases, detergents, chelating agents, and solvents. If necessary, these can be removed before amplification by common DNA extraction techniques. Genetic stability of target sequences has been addressed by developing primer sets which bind to evolutionarily-conserved, yet species-specific sequences within the genes for ribosomal RNA. Furthermore, use of ultraviolet radiation to covalently cross-link any potentially contaminating DNA in vessels and reagents, and on equipment used in the PCR and LCR protocols, has helped reduce false-positive amplifications. Finally, the potential for contamination can be monitored by performing all steps in the amplification protocols with mixtures to which no known source of templet DNA has been added. ___________________________________ SUMMARY In summary, variability and uncertainty with currently available serological techniques for rapid laboratory confirmation of Lyme disease limit the utility of such tests. Such uncertainty compounds the difficulty of physicians to either confirm or rule out Lyme disease in patients with symptoms and histories consistent with infection by Lyme spirochetes. Questions surrounding the ability to accurately diagnose Lyme disease have been cited as reasons behind delayed or inappropriate treatment of patients, and in disallowance of insurance coverage for treatment. During the last few years, several new approaches for confirming infections have been investigated and developed. These include detection of spirochetal antigens by T-cell proliferative assays, antibody-based capture and detection assays, and detection of spirochetal nucleic acids by enzyme chain reactions. Continued development and evaluation of these techniques may provide effective tools to facilitate monitoring infections by Lyme spirochetes in arthropod and mammalian hosts. __________________________________ ACKNOWLEDGMENTS Special thanks go to Ronald Harbeck, Leonard Mayer, and Roxanne Shively for critical review of this article, and to Betty Kester for help in preparing the article. __________________________________ REFERENCES 1. Burgdorfer, W., et al. 1982. "Lyme disease--a tick-borne spirochetosis?" Science 216:1317-1319. 2. Baranton, G., et al. 1992 "Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis." Int. J. Syst. Bacteriol. 42:378-383. 3. Steere, A. C. 1989. "Lyme disease." New. Engl. J. Med. 321:586-596. 4. Szczepanski, A., and J. L. Benach. 1991. "Lyme borreliosis: host responses to Borrelia burgdorferi." Microbiol. Rev. 55:21-34. 5. Callister, S. M., et al. 1991. "Lyme disease assay which detects killed Borrelia burgdorferi." J. Clin. Microbiol. 29:1773-1776. 6. Dressler, F. el al. 1991. "The T-cell proliferative assay in the diagnosis of Lyme disease." Ann. Int. Med. 115:533-539. 7. Hyde, F. W., et al. 1989. "Detection of antigens in urine of mice and humans infected with Borrelia burgdorferi, etiologic agent of Lyme disease." J. Clin. Microbiol. 27:58-61. 8. Dorward, D. W., et al. 1991. "Immune capture and detection of extracellular Borrelia burgdorferi antigens in fluids and tissues of ticks, mice, dogs, and humans." J. Clin. Microbiol. 29:1162-1171. 9. Dorward, D. W., and C. F. Garon. Unpublished data. 10. Schwartz, J., et al. 1991. "Determination of Borrelia burgdorferi infection rates in Ixodes dammini ticks by three methods." Abstracts of the General Meeting of the American Society for Microbiology 91:80. 11. Rosa, P. A., and T. G. Schwan. 1989. "A specific and sensitive assay for the Lyme disease spirochete Borrelia burgdorferi using the polymerase chain reaction." J. Infect. Dis. 160:1018-1029. 12. Persing, D. H., et al. 1990. "Detection of Borrelia burgdorferi DNA in museum specimens of Ixodes dammini ticks." Science 249:1420-1423. 13. Goodman, J. L., et al. 1991. "Molecular detection of persistent Borrelia burgdorferi infection in the urine of patients with active Lyme disease." Infect. Immun. 59:269- 278. 14. Hu, H., et al. 1991. "Detection of Borrelia burgdorferi by ligase chain reaction." Abstracts of the General Meeting of the American Society for Microbiology. 91:79. 15. Marconi, R. T., and C. F. Garon. 1992. "Development of polymerase chain reaction primer sets for diagnosis of Lyme disease and for species-specific identification of Lyme disease isolates by 16S rRNA signature nucleotide analysis." J. Clin. Microbiol. 30:2830-2834 ----------------------------------------------------------------------------- The Health Info-Com Medical Newsletter is distributed every 2 weeks and contains MMWR reports, FDA news, medical research articles and AIDS news. To subscribe, users should send e-mail to: [email protected] (or [email protected]) In the first line of the memo, write: sub mednews FirstName LastName --- send questions to [email protected] (David Dodell) --- ----------------------------------------------------------------------------- III. ***** JARGON INDEX ***** Bb - Borrelia burgdorferi - The scientific name for the LD bacterium. CDC - Centers for Disease Control - Federal agency in charge of tracking diseases and programs to prevent them. CNS - Central Nervous System. ELISA - Enzyme-linked Immunosorbent Assays - Common antibody test EM - Erythema Migrans - The name of the "bull's eye" rash that appears in ~60% of the patients early in the infection. IFA - Indirect Fluorescent Antibody - Common antibody test. LD - Common abbreviation for Lyme Disease. NIH - National Institutes of Health - Federal agency that conducts medical research and issues grants to research interests. PCR - Polymerase Chain Reaction - A new test that detects the DNA sequence of the microbe in question. Currently being tested for use in detecting LD, TB, and AIDS. Spirochete - The LD bacterium. It's given this name due to it's spiral shape. Western Blot - A more precise antibody test. IV. ***** HOW TO SUBSCRIBE, CONTRIBUTE AND GET BACK ISSUES ***** SUBSCRIPTIONS: Anyone with an Internet address may subscribe. Send a memo to [email protected] in the body, type: subscribe LymeNet-L <Your Real Name> DELETIONS: Send a memo to [email protected] in the body, type: unsubscribe LymeNet-L CONTRIBUTIONS: Send all contributions to [email protected] All are encouraged to submit questions, news items and commentaries, regardless of expertise. 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