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Show Joumal" f ClI";..,, l Ncun1- 0l'htIItl1l1ll11ogli ge): 76- 78, 1989. Editorial Comment © 1989 Raven Press, Ltd., New York Diagnostic Detente and Spirochetal Serology Zierhut and colleagues retrace their steps through a diagnostic labyrinth in search of an etiologic diagnosis for a case of panuveitis. Their diagnostic efforts are complicated by the fact that a physician who previously attended the patient chose to wield the double- edged sword of empiric corticosteroid therapy without first excluding infection as a probable cause of the patient's symptoms. Zierhut's group explores several layers of laboratory evidence that alternately implicate either syphilis or borreliosis as possible etiologies for the patien t' s disease. In the spirit of the clinicopathological conference, I shall reexamine the evidence, offer my own diagnosis, and discuss some possible shortcomings of the various laboratory diagnostic tools that have been applied to this case. The patient's serum contained antibodies that reacted in a nontreponemal syphilis serology test ( a Venereal Disease Research Laboratory ( VDRL) test was 3 + reactive at a 1120 dilution) and antibodies that reacted to treponemaI syphilis serology methods ( Treponema pallidum hemagglutination assay ( TPHA) was reactive at 1110,240 dilution, and fluorescent treponemaI antibody absorption test ( FTA- ABS) was reactive at 1116 dilution). Serologic tests for Lyme borreliosis ( LB) yielded reactive results at one laboratory and conflicting reactive and nonreactive results at a second laboratory. The immunoblot ( Western blot) test failed to implicate either spirochete. In the end, it was the patient's intervention that prevailed, when " ... the patient denied having had a syphilitic infection." Borreliosis provided a nonstigmatized diagnosis for both the patient and for her physicians. I believe that the patient's clinical and laboratory profile support the diagnosis of syphilis. Although serum samples from patients with LB may crossreact in the FTA- ABS test at a low titer, LB serum samples are nonreactive in the VDRL test. The magnitude of thE' TPHA titer felr exceeds the cross- ; '" r, ""' r bl'(' n repllrted ior LB 76 Serology tests have their shortcomings and were never intended to replace clinical judgment in the diagnosis of disease. The technical shortcomings of serology tests as tools for the diagnosis of LB have been introduced in this journal ( 1) and have been recently reviewed by Barbour ( 2). A concise restatement of some working principles is summarized as follows: Negative serologic findings for LB do not exclude active LB. Some patients with seropositive LB produce a family of antibodies that will crossreact with some treponemal antigens in the FTA- ABS test but that fail to crossreact in the VDRL test. Some patients with a Treponema pallidum infection produce a family of antibodies that crossreact in LB serology methods. Reversion from a seropositive to a seronegative state following antibiotic therapy should not be expected in every case of LB. It is puzzling that LB may show clinical progression in some hosts despite the presence of very high levels of antibodies in the blood or cerebrospinal fluid. This suggests that some classes of antibody are not able to protect the host against the pathologic effects of the spirochete. A second issue requiring further study is the observation that certain experimental animals may harbor large numbers of the spirochete and suffer no obvious tissue injury. Immunoglobulin M- class antibodies are generally presumed to indicate early infection. Phenomena that are discussed at Lyme disease symposia but are rarely addressed in print are isolated reports of the appearance of a delayed onset of peak IgM level in chronic cases of LB after a welldeveloped IgG response has appeared. A satisfactory explanation for this phenomenon has not been found. An observation from relapsing fever borreliosis may provide a clue to the understanding of the later- appearing IgM peaks. The relapsing fever borreliae evade the humoral response of the host by modulating the structure of selected EDITORIAL COMMENT 77 proteins that appear on the spirochete's surface. This structural change is due to the synthesis of new classes of proteins orchestrated by gene rearrangements within the spirochete. Antibodies that the host animal produces are directed against obsolete surface protein epitopes and are therefore unable to bind to the redesigned spirochete. The host with relapsing fever borreliosis continues to produce generation after generation of antibodies against the newly synthesized spirochetal proteins. Perhaps we may be observing an analogous situation in some cases of LB with late- onset IgM level peaks. lmmunoblotting techniques ( Western blots) are research methods that offer an anatomic dissection of the humoral immune response to the proteins of a pathogenic microbe. Detergents reduce the spirochete to a solution of proteins. Electrophoresis sorts the proteins according to their molecular weight. The ladder- like array of sorted proteins is transferred by direct apposition ( blotting) to a nitrocellulose membrane that permanently fixes each of the bands on the membrane in a position that exactly corresponds to the position that the band occupied on the electrophoresis gel. Serum is layered over the membrane. Antibodies that correspond to specific protein epitopes are confined to discrete bands on the blot. In this technique, a titer is converted to an analog signal. The multiplicity of antibodies that recognized specific proteins can be visualized, quantitated, and assigned IgM and IgG classes, and a pattern of early and late- appearing antibodies can be studied. The only consistent pattern that has emerged from Western blot studies of sequential serum specimens is the reverse of the predicted sequence of antibody production. The first antibody to appear is directed against the flagellar structural proteins that are confined beneath the first of two concentric cylinders that form the spirochete. Antibodies to the proteins of the outer surface of the spirochete appear later or not at all. It is unfortunate that the flagellar antibodies are not specific for LB infection. All spirochetes have flagellae, and the flagellar proteins of Borrelia burgdorferi crossreact with them. The paradox of the flagellar proteins has played itself out in the design of serology test reagents at two reference laboratories. The flagellar proteins of B. burgdorferi have been manipulated to increase the sensitivity and specificity of LB serology methods. At one reference laboratory, the flagellar antigens were intentionally deleted from the other spirochetal antigens in the manufacture of an enzyme- linked immunosorbent assay ( ELISA) plate. This step was based on the premise that an ELISA without flagellar epitopes would have fewer false positive results by eliminating the possibility of detection of antibodies that might react with flagellar proteins of unrelated spirochetes. A second reference laboratory designed an ELISA plate that included the flagellar proteins. This was based on the premise that because the earliest antibody response to LB infection is directed against the flagellar proteins, sensitivity and specificity would be enhanced. It is not clear whether either institution has found the " best" ELISA antigen formulation. Experiments with sorbent in LB serology tests have yielded discrepant results. The impetus to try sorbent came from the FTA- ABS procedure. The sorbents used so far have diminished the titers of reactive LB serum samples in parallel with the titers of crossreacting serum samples. For this reason, most reference laboratories do not use sorbent in LB serology methods. Serology titers cannot be compared between laboratories because the reagents are not prepared according to an international standard. Several strains of the spirochete are used in reagent manufacture. The protein composition of wild strains of LB spirochetes is not uniform, and the protein composition of laboratory- adapted strains may change as they are maintained in the laboratory. Sequential analysis of serum specimens from the same patient by one laboratory over a protracted period should be compared only qualitatively ( i. e., as reactive, borderline, or nonreactive) and should never be compared quantitatively. This is because the threshold [ ELISA or indirect immunofluorescence assay ( IFA)] for a reactive specimen fluctuates from day to day. If a quantitative comparison is required for sequential serum samples from one patient, all specimens should be frozen as they are obtained and submitted as a group for processing by the reference laboratory. Various modifications in the method of antibody detection have resulted in a proliferation of LB serology methods. The IFA, ELISA, and flAX methods have received U. S. Food and Drug Administration ( FDA) approval for the diagnosis of LB. Hemagglutination inhibition and automated fluorescence techniques are under FDA review and are expected to be approved soon. All antibody detection methods require meticulous attention to detail and careful quality control to maintain specificity and sensitivity of testing. Some extravagant claims are made from time to time that a new method has rendered all other methods of antibody detection obsolete. A physician should avoid the rhetoric and focus attention on the quality of the antigen I Gin Neuro- ophthalmol, Vol. 9, No. 2, 1989 78 EDITORIAL COMMENT mixture that is used in a specific method. It is also worth\ vhile to periodically review the laboratory's performance in proficiency testing programs. New York has recently implemented a series of mandatorY proficiencv testing programs for LB serology. General regulation of laboratories offering LB antibodv detection is expected to follow on a national scope in the near future. Problem serum samples are defined by me as those that react in only one of the two common antibody detection methods ( IFA or ELISA). In all clinical respects, patients with problem serum samples resemble patients with active LB who have seropositive results in both the IFA and ELISA. It seems prudent to consider retesting patients with clinical symptoms of LB by the complementary method ( IFA or ELISA) if a single method yields a seronegative result. Dr. J. Lawton Smith has recommended a four- part spirochetal serology panel ( VDRL, FTA- ABS, IFA- LB and ELISA- LB) because this combination of tests offers broadspectrum coverage of antibodies that might fail to be detected if only one serology method is used. A seronegative state in late spirochetal disease was first defined for syphilis by Dr. Smith 25 years ago and was further codified in his monograph Spirochetes in late seronegatipt! syphilis, penicillin notwithstanding ( 3). Dr. Smith's definition of seronegative syphilis in 1969 included three criteria that physicians in practice in 1988 should commit to memory: " 1. A patient showing clinical signs or symptoms of late syphilis. 2. A nonreactive serum reagin test ( as the VDRL test) 3. A reactive specific treponemal test ( as the [ T. pallidum immobilization test] TPI or FTA- ABS test) and/ or demonstration of the spirochete." / ClilI Neuro- ophthalmol. Vol. 9, No. 2, 1989 True seronegativity in LB has recently been defined by Dattwyler and associates ( 4). They have identified 17 patients with clinically persistent and progressive Lyme disease whose antibody response to the infection was abrogated by treatment with oral antibiotics early in their disease. A proliferative reaction of host lymphocytes in response to in vitro coincubation of lymphocytes with B. burgdorferi was the sole evidence for active LB infection. Dattwyler's group has demonstrated that immunologic memory of exposure to the spirochete resides only in the cellular arm of the immune system in certain patients with active and progressive spirochetal infection. Diagnosis would be very much simpler if pathogenic spirochetes could be cultivated from blood, body fluids, or tissue. A reliable culture and sensitivity tool is unlikely to appear for LB and may never be realized for T. pallidum. Lyme borreliosis and syphilis are increasing at alarming rates in North America and on other continents. It is unethical to withhold antibiotic treatment when active infection with either of these spirochetal pathogens is recognized. It is equally deplorable to avoid confronting the problems that are inherent in the diagnosis of these spirochetal infections. Alan B. MacDonald, M. D. Southampton Hospital Southampton, New York REFERENCES 1. MacDonald AB. Ambiguous serologies in active Lyme borreliosis. JClin Neuro Ophthalmol 1988; 8: 79-- 80. 2. Barbour AG. Laboratory aspects of Lyme borreliosis. elin Microbiol Rev 1988; 1: 399- 414. 3. Smith JL. Spirochetes in late seronegative syphilis, penicillin notwithstanding. Springfield, Illinois: Charles C Thomas Publisher, 1969. 4. Dattwyler RJ. Volkman OJ. Luft BJ. Seronegative Lyme disease. N Engl JMed 1988; 319: 1441-{'. |