Wisconsin proficiency program for Lyme disease: A program for improving detection of antibodies to Borrelia burgdorferi

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luteinizing hormone. FASEB J 3:A1474. 16. Weigent DA, Baxter JB, Wear WE, et al.: Production of immunoreactive growth hormone by mononuclear leukocytes. FASEB J 2:2812-8, 1988. 17. Kao T-L, Keenan BS, Meyer WJ: Char-

acterization of human growth hormone secreted by lymphocytes. Endocrine Society Annual Meeting Abstract, 1990. 18. Hiestand PC, Mekler P, Nordmann R, et al.: Prolactin as a modulator of lymphocyte responsiveness provides a possible

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mechanism of action for cyclosporin. Proc Natl Acad Sci USA 83:2599-603, 1986. 19. Marwick AJ, Lolait SJ, Funder JW: Immunoreactive arginine vasopressin in the rat thymus. Endocrinology 119:1690-6, 1986.

Wieconoln Proficiency Program for Lyme Disease: A Program for Improving Detection of A n t i b o d i e s t o Be555rli4 JpurfpJe f ri Lori L. Bakken, Steven M. Callister, Kay L. Case, and Ronald F. Schell Wisconsin State Laboratory of Hygiene, Madison, Wisconsin, and Gundersen Medical Foundation and Lutheran Hospital, La Crosse, Wisconsin

B

orrelia burgdorferi, the

spirochete that causes Lyme disease, can p infect many nssues of the body including the skin, heart, nervous system, and joints.17 Because of this, Lyme disease symptoms can resemble those of other illnesses such as influenza, aseptic meningitis, multiple sclerosis, or rheumatoid arthritis. This ability to cause symptoms similar to other diseases can make the accurate diagnosis of Lyme disease difficult. Therefore, the physician relies heavily on the ability of the laboratory to detect accurately antibodies to B. burg-

dorferi. Serologic testing is currently the only practical method for establishing a Lyme disease diagnosis. Infected humans produce IgM and IgG antibodies that recognize B. burgdorferi antigens, lgM antibodies generally peak three to six weeks following infection, while IgG antibodies peak one to three weeks later. The concentration of IgG antibody may then remain elevated for weeks to years following infection. 4' 16 Western immunoblotting is used by some laboratories to detect antibodies to individual antigenic components of B, burgdorferi. However, its use as a practical clinical laboratory test remains unclear. For example, some investigators have shown that this test is superior to the indirect ELISA for determining early Lyme disease, 7 while others have demonstrated that it is not the method of choice 0197-1859/91/$0.00

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for diagnosing early Lyme disease. 2'9 Until these concerns are resolved, this procedure appears to have limited usefulness in most routine diagnostic laboratories. Currently, indirect immunofluorescent assays (IFAs) and enzyme-linked immunosorbent assays (ELISAs) are the most commonly used methods for identifying and quantifying the presence of antibodies to B. burgdorferi. Some investigators have reported that ELISA is more sensitive than IFA, 7 while others report that IFA is as predictive as ELISA if the laboratory has experience and conservative cutoff titers are used.12'13 Regardless, both systems remain somewhat less than perfect because of both common and unique problems associated with each. Whole borrelial cells are invariably used as the antigen in IFA. However, this is where similarities among IFA test procedures end since neither the antigens nor the methods have been standardized among laboratories. For example, diagnostic titers considered indicative of B. burgdorferi exposure vary among laboratories depending in part on their individual criterion for intensity of fluorescence. This causes significant cutoff points to vary.

Using ELISA, which generally utilizes either whole cells or sonicated cells as antigen, sera are considered diagnostically positive if they elicit absorbances 2.5 to 3 standard deviations above the mean abI © 1991 Elsevier Science Publishing Co., Inc.

sorbance of a group of healthy controls. This technique has also not been standardized among laboratories and wide variations currently exist. The cultures of B. burgdorferi used as antigen may also cause differences among test procedures. We have observed up to 16-fold differences in reported IgM antibody levels of individual patient serum when performing IFA with diagnostic slides prepared using B. burgdorferi cultured in separate BSK medium containing different lots of fraction V bovine serum albumin. 3 It is easy to envision the same effect occurring with ELISA. Another variable affecting serologic testing may be use of other B. burgdorferi isolates as antigen. Most laboratories and commercial companies use the original Shelter Island, NY, USA, isolate B31 (ATCC 35210). However, other isolates are also used. Greene et al. 6 showed that immunoblot patterns of sera from dogs naturally infected with B. burgdorferi were changed depending on which isolate of B. burgdorferi was used. We have also shown that different isolates significantly change immunoblot patterns of sera from humans with Lyme disease. 18 In addition, Schwan et al. 14 demonstrated that noninfectious B. burgdorferi isolate B31 (multiple passage) had a reduced immunoblot compared to an infectious isolate. These observations provide evidence that a serum may be falsely labeled negative if reactivity varies depending on I

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the B. burgdorferi isolate or its passage. Even after IgM or IgG antibody determination procedures are successfully standardized, both sensitivity and specificity will likely remain troublesome. While patients with Lyme disease almost always have elevated IgG antibody titers, significant IgG titers are present in only 5060% of Lyme cases during the first six weeks of infection.l'4 Because IgG antibodies are not often detected during early infection, IgM antibody determinations may be useful. However, IgM antibodies are generally less specific than IgG antibodies and false-positive results are relatively common in healthy patients and those with other disease syndromes such as syphilis, Rocky Mountain spotted fever, autoimmune diseases, and neurologic disorders. 5"7 Antibiotic therapy has also been shown to blunt the immunologic response so that diagnostic thresholds are never reached. 1o These factors have engendered strong feelings about the reliability of Lyme serology. In fact, several researchers have demonstrated that serology is not reliable for diagnosing Lyme disease. For example, Schwartz et al. t5 found little agreement among four laboratories that received sera from 132 outdoor workers. Likewise, Hedberg et al. 8 reported several significant discrepancies between test results on identical samples. After observing considerable interlaboratory variability, Luger and Krauss 11 concluded that the sensitivity and specificity of Lyme disease assays needs improvement and that a qualtiy assurance program is also needed so that performance of individual laboratories can be monitored.

Wisconsin Proficiency Testing Program The Wisconsin State Laboratory of Hygiene in Madison and the Gundersen Medical Foundation and Lutheran Hospital in La Crosse, Wisconsin, have introduced a'~oficiency testing program designed to allow individual laboratories and manufacturers of serologic kits to evaluate the quality of their Lyme disease assays. During 1990, three normal and six case-defined Lyme disease sera were sent to 71 laboratories for evaluation. Triplicate samples of each unidentified serum were randomized and distributed

TABLE 1. RECOMMENDATIONS 1. Laboratory personnel must pay special attention to their quality control procedures. 2. Laboratory personnel should develop internal controls in addition to controls provided with commercial kits. 3. Laboratory personnel should choose a negative, low positive, and a high positive serum to run as controls. 4. Laboratory personnel should test internal controls, in duplicate, with each run. 5. Control sera should be aliquoted and frozen at - 20°C or less. Aliquots should be held at 4°C when in use for no longer than 2 weeks. 6. Laboratory personnel should use vials that have airtight seals to prevent evaporation of s e r a .

during four mailings so that intra- and interlaboratory results could be compared within and across individual mailings. The performance of each laboratory was evaluated by determining their ability to detect accurately the presence or absence of antibodies to B. burgdorferi. In addition, the ability to reproduce results among triplicate serum samples was evaluated. Several significant trends became apparent. Sensitivity among laboratories was highly variable. Generally, Lyme disease sera which contained high levels of IgG (/> 1:512) were consistently identified, although some laboratories failed to correctly identify these positive sera. Accurate detection of antibodies to B. burgdorferi using IFA and ELISA was dependent on the level of antibodies in the serum. Accuracy decreased significantly when lesser amounts of antibody to B. burgdorferi were present. As expected, the specificity of the Lyme disease serologic assays remained troublesome. Serum from non-case-defined individuals that did not contain detectable antibodies to B. burgdorferi organisms were often (approximately 16%) identified as positive for Lyme disease. Interestingly, ELISAs were less specific than IFAs. Most disconcerting was the inability of many laboratories to consistently detect levels of antibodies to B. burgdorferi organisms when assaying triplicate samples of an individual serum. When serum contained high levels of antibodies to B. burgdorferi, Lyme disease assays were generally reproducible. However, the ability of the participating laboratories to detect the same levels of antibodies to B. burgdorferi after triplicate testing decreased significantly as the antibody level in the serum was reduced. Laboratories © 1991 Elsevier Science Publishing Co., Inc.

using ELISA produced more reproducible results than those using IFA, although reproducibility was still dependent on the level of B. burgdorferi antibody present. This was likely because ELISAs are generally interpreted using automated instruments, while IFA interpretations are subjective. Lyme disease has recently emerged as a new infection with worldwide significance. As a consequence, it becomes increasingly important to diagnose it accurately. The varied presentations attributable to this disease may make this task formidable. In these instances, diagnostic laboratory testing becomes an invaluable aid. However, our results to date illustrate problems that continue to plague Lyme serology. The Wisconsin Lyme disease proficiency testing program is enabling the extent of these problems to be determined. These results also demonstrate the need for a program which determines the accuracy of individual diagnostic Lyme disease assays. Using these preliminary data, we have begun to formulate recommendations which should improve the quality of Lyme disease serology (Table 1). In addition, we are expanding this program so that laboratories will have a means to monitor assay performance and insure optimum accuracy and reproducibility, cIN

References 1. Ackermann R, Kahatski J, Boisten HP, et al.: Ixodes ricinus spirochete and European erythema chronicum migrans disease. Yale J Biol Med 57:573-80, 1984. 2. Berardi VP, Weeks KE, Steere AC: Serodiagnosis of early Lyme disease: Analysis of IgM and IgG antibody responses by 0197-1859/91/$0.00 + 2.20

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using an antibody-capture enzyme immunoassay. J Infect Dis 148:754-60, 1988. Callister SM, Case KL, Agger WA, et al.: Effects of bovine serum albumin on the ability of Barbour-Stoenner-Kelly medium to detect Borrelia burgdorferi. J Clin Microbiol 28:363-5, 1990. Craft JE, Grodzicki RL, Steere AC: Antibody response in Lyme disease: Evaluation of diagnostic tests. J Infect Dis 149:789-95, 1984. Duffy J, Mertz LE, Wobig GH, et al.: Diagnosing Lyme disease: The contribution of serologic testing. Mayo Clin Proc 63:1116--21, 1988. Greene RT, Walker RL, Burgess EC, et al.: Heterogeneity in immunoblot patterns obtained by using four strains of Borrelia burgdorferi and sera from naturally exposed dogs. J Clin Microbiol 26:2287-91, 1988. Grodzicki RL, Steere AC: Comparison of immunoblotting and indirect enzymelinked immunosorbent assay using different antigen preparations for diagnosing early Lyme disease. J Infect Dis 157:790-

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7, 1988. 8. Hedberg CW, Osterholm MT, MacDonald KL, et al.: An interlaboratory study of antibody to Borrelia burgdorferi. J Infect Dis 155:1325-7, 1987. 9. Karlsson M, Mollegard 1, Stiernstedt G, et al.: Comparison of Western blot and enzyme-linked immunosorbent assay for diagnosis of Lyme disease. Eur J Clin Microbiol 8:871-7, 1989. 10. Liu NY, Dinerman H, Levin RE, et al.: Randomized trial of doxycycline vs amoxicillin/probenecid for the treatment of Lyme arthritis: Treatment of non-responders with IV penicillin or ceftriaxone (abst.). Arthritis Rheum 32:$46, 1989. 11. Luger SW, Krauss E: Serologic tests for Lyme disease: lnterlaboratory variability. Arch Intern Med 150:761-3, 1990. 12. Magnarelli LA, Meegan JM, Anderson JF, et al.: Comparison of an indirect fluorescent-antibody test with an enzymelinked immunosorbent assay for serological studies of Lyme disease. J Clin Microbiol 20:181-4, 1984. 13. Russell H, Sampson JS, Schmid GP, et

al.: Enzyme-linked immunosorbent assay and indirect immunofluorescence assay for Lyme disease. J Infect Dis 149:46570, 1984. Schwan TG, Kine KK, Schrumpf ME, et al.: Antibody response in white-footed mice (Peromyscus/eucopus) experimentally infected with the Lyme disease spirochete (Borre/ia burgdorferi). Infect Immun 57:3445-51, 1989. Schwartz BS, Goldstein MD, Ribeiro JM: Antibody testing in Lyme disease, a comparison of results in four laboratories. JAMA 262:3431-4, 1989. Steere AC, Grodzicki RL, Kornblatt AN, et al.: The spirochetal etiology of Lyme disease. N Engl J Med 308:733-40, 1983. Steere AC, Malawista SE, Bartenhagen NH, et al.: The clinical spectrum and treatment of Lyme disease. Yale J Biol Med 57:453~1, 1984. West RM, Case KL: Effects of different Borrelia burgdorferi isolates on the Lyme disease Western blot (abst). General Meeting of the American Society for Microbiology, Dallas, TX, 1991.

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Clinical Immunology Newsletter, Volume 11, 1991 Index academic stress, 7:102-103 acetylcholine receptor antibody, 11:165 ACTH, see corticotropin activated partial thromboplastin time, and lupus anticoagulant, 6:92-93 acute phase response, 9:129 mechanisms involved in and biological and clinical significance, 9:129-132 agammaglobulinemia congenital, 5:67-68 X-linked (Bruton's), 10:147 agarose gel electrophoresis, in acute and inflammatory states, 9:132-135 alpha2 macroglobulin, 9:133, 134 aneuploidy, DNA, 4:51, 54 anti-LC-1, 11:164 anti-liver-kidneymicrosome antibody, 11:163-164 anti-retroviral therapy, cellular immunity changes resulting from, 5:76 anti T-cell receptor therapy, 11:172 antibodies antiphospholipid, see antiphospholipid antibodies deficiency, intravenous immune globulin therapy for, 10:151 detection, with antigen-coated microspheres, 4:61-62 EBV-specific, 6:82 human anti-mouse, 4:62 polyclonal, 9:136 quantification, fluorescence quantification versus, 4:53

response to Borellia burgdorferi, 8:114-115 see also autoantibodies anticardiolipin ELISA test, 3:33, 34-37 anticardiolipin kits, 3:41-47 anticoagulants, lupus, 6:91-96 anticytoskeleton antibodies, 11:163 antigen Borrelia burgdorferi, 8:115 carcinoembryonic, 4:63 E B V , 6:82-83 HIV, 5:75-76 liver, 11:164 luekocyte differentiation, 4:51 non-HIV, 5:65-66 syphilis, 8:125-126 tolerance therapies, 11:171-172 antigen-capture assays, 4:62-63 antigen-coated microspheres, antibody detection with, 4:61-62 antimitochondrial antibodies, 11:163, 164 antiphospholipid antibodies, 10:154 immunochemistry of, 3:33, 38-41, 41-47 measurement techniques, anticardiolipin kits, 3:41--47 specificity, 3:38-40 antipbospholipid syndrome, 3:33 antismooth muscle antibodies, 11:163 arginine vasaopressin, production by immune system, 12:183, 185 arthritis, Lyme, 8:120-121 asialoglycoprotein receptor (ASGPR) antibody, 11:165

asthma, intravenous immune globulin therapy in, 10:154 Augustine JA, 2:17, 18-27 autoantibodies in autoimmune chronic active hepatitis, 11:161, 1 6 2 - I 67 striational, 11:161,167-170 autoimmune chronic active hepatitis diagnostic significance of autoantibodies in, 11:161,162-167 subgroups of, 11:163 autoimmune disorders, intravenous immune globulin therapy for, 10:151,153-154 autoimmunity, T cell, 11:170-175

B-cell malignancies, infection prevention in, intravenous immune globulin therapy for, 10:152 Bakken LL, 12:186-188 bead-based assays, application for flow cytometry analysis, 4:60--64 Bines SD, 4:49, 54-58 biologic response modifiers, 2:30 Birx DL, 10:145, 150-157 bone marrow transplantation, infection prevention in, intravenous immune globulin therapy for, 10:152 Borrelia burgdorferi,in Lyme disease antibodies to, improving detection of, 12:186188 cellular immune response to, 8:113, 118-124 •

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