Clinical Microbiology Newsletter Vol. 21, No. 16 August 15, 1999

The Impact of Vaccination against Lyme Borreliosis on Laboratory Serodiagnosis Steven M. Callister, Ph.D. Dean A. Jobe, M.S. Section of infectious Diseases and

Microbiology Research Laboratory Gundersen Lutheran Medical Center La Crvsse, Wisconsin 54601

Ronald F. Schell, Ph.D. Wisconsin State Laboratory of Hygiene Department of Medical Microbiology

and Immunology University of Wisconsin Madison, Wisconsin 53706

Introduction Lyme borreliosis, a multisystem

illness caused by transmission of Borrelia burgdogeri sensu lato (Bb) from Zxodes spp. ticks, is a relatively common vector-borne disease world- wide (1). In the United States, most cases of Lyme borreliosis occur in the northeast and upper midwest, although cases have been reported nationwide. From 1991 to 1996, approximately 70,000 cases were reported in the United States (2). The widespread occurrence and increasing numbers of Lyme borreliosis cases have provided strong impetus for developing an effec- tive vaccine. Recently, clinical trials of two Lyme borreliosis vaccines based on outer surface protein (0sp)A demon- strated that they could protect humans from becoming infected with Bb (3,4). These results prompted the Food and Drug Administration (FDA) to approve an OspA Lyme borreliosis vaccine for use in 15 to 70 year-old individuals.

Approval of a vaccine is an important step toward preventing Lyme borreliosis. Vaccination of susceptible individuals in areas where Lyme borreliosis is

endemic should significantly reduce the morbidity associated with this illness. However, the vaccine will not entirely eliminate the risk of becoming infected with Bb even after repeated vaccina- tions. The OspA vaccine was less than 50% effective in preventing infection with Bb after two injections and only 78% effective after a third injection (4). In addition, the duration of protective immunity (5) and the number of sub- sequent yearly booster vaccinations required to maintain sustained high levels of protective borreliacidal anti- bodies remain unknown. Antigenically variant strains of Bb are also found in the United States (6) and infection with these spirochetes could still occur after vaccination. These shortcomings make it probable that OspA vaccinees may still become infected with Bb. In fact, Bb infections have been demonstrated in a human (7), dogs (8), and rabbits (9) despite vaccination.

Serodiagnosis of Lyme BorreliQsis Diagnosis of Lyme borreliosis is

based upon clinical symptoms, history of exposure in an endemic area, and laboratory test results. In ambiguous cases, there is a greater reliance on lab- oratory tests to confirm the diagnosis. Serologic testing offers the most practi- cal means for confirming Lyme borre- liosis. Shortly after the discovery of Lyme borreliosis, however, several investigators demonstrated poor intra- and interlaboratory agreement of sero- logic results (lo- 12). The poor perfor- mance of serologic testing combined with inappropriate and excessive use of these tests (13- 15) made it difficult to

interpret test results. Thus, researchers have also spent considerable effort devel- oping more accurate serodiagnostic testing procedures. To date, a single sensitive and highly specific laboratory test for detecting Lyme borreliosis has not become widely available. During the past several years, a two-tiered approach for serodiagnosing Lyme borreliosis has been advocated by the Centers for Disease Control and Prevention (CDC) as a means to mini- mize the numbers of false-positive and false-negative serologic results (16).

In This Issue

The Impact of Vaccination against Lyme Borreliosis on Laboratory Serodiagnosis . . . . . 129 This article is a brief review of he laboratory diagnosis of Lyme Disease and the efforts to develop a viable vaccine. The problems related to Western blot conjirmation and serologic false positives due to the presence of antibodies to the vaccine are discussed. A new conjirmarory test of borreliacidal antibody is described as an option in early disease.

Salmonella hadar Pneumonia in a Heart lkansplant Patient.. .133

a case report

Bacteroides fragilis Arthritis of the Knee with Fatal Outcome . . . . . . . . . . . . . . . . . . . .134

a case report

Clinical Microbiology Newsletter 21: 16.1999 8 1999 Elsevier Science Inc. 0196-4399/99 (see frontmatter) 129

The accuracy of the two-tiered test- ing approach is dependent on a sensitive screening test and a specific confirma- tory test. The most common screening tests are the indirect immunofluores- cence assay (IFA) and the indirect enzyme-linked immunosorbent assay (ELISA). Anti-Bb antibodies are cap- tured by whole-cell lysates, sonicated extracts, or partially purified Bb. Quantitative results are expressed as bound total immunoglobulin (Ig), IgM, or IgG. Many laboratories prefer the ELISA because it is less subjective and better suited for large workloads. Cross reactivity with antibodies against other microorganisms and false-positive results from individuals with various autoimmune and viral syndromes cause these tests to have high sensitivity but a significant lack of specificity. Because of this, equivocal or positive results are confirmed by using a more specific test such as Western blot (WB). WI3 increases the specificity by allowing antibodies against individual proteins to be detected and analyzed. An IgM WB is considered positive if 2 of the 24, 39, or 41 kDa bands are present. An IgG WB is considered positive if 5 of the 18,21,28,30, 39,41,45,58,66, or 93 kDa bands are present (16).

Several investigators have demon- strated accurate results using the two- tiered approach (17-19). However, the accuracy of this approach is dependent on testing patients with a high pretest probability of having Lyme borreliosis (20,21). A significant number of false positive results have been observed in settings where the probability of con- tracting Lyme borreliosis is low. The predictive values of IgM Western blots is less than 50% when the pretest proba- bility of Lyme borreliosis is less than

10% (19,22). Requests for serodiagnostic testing

for Lyme borreliosis have increased dramatically during the past several years. Market analysts estimated that 2.79 million tests for Lyme borreliosis were performed in 1995 (23). Perform- ing !arge numbers of tests to identify relatively small numbers of Lyme bor- reliosis cases makes it certain that tests are often performed in patients with low pretest probabilities including indivi- duals in highly endemic areas with non- specific signs and symptoms such as headache, fatigue, myalgia, or arthralgia. Thus, it is not surprising that inaccurate testing results continue to confound Lyme borreliosis serodiagnosis despite reports demonstrating the ability of the two-tiered system to accurately detect Lyme borreliosis (17- 19).

In 1991, Bakken et al. (12) demon- strated the poor accuracy and reliability of serodiagnostic testing by different laboratories. Fifty-five percent of the laboratories could not accurately identify serum samples containing antibody against Bb. In addition, 2 to 7% of labo- ratories identified serum samples from individuals with no known exposure to Bb as positive. There was also a strik- ing inability of many laboratories to reproduce results from split samples from the same individual. In a more recent study, Bakken et al. (24) showed that the sensitivity and specificity of screening tests for Lyme borreliosis had not improved from 1992 through 1994. The specificity of the Lyme borreliosis assays decreased from approximately 95% to approximately 8 1% during the three-year period. False-positive test results approached 55% with some of the serum samples from healthy donors. In addition, the sensitivity fluctuated

between 93 and 75% depending upon the conjugate used by the laboratories.

Effect of Vaccination on Serodiagnosis

Serodiagnosis by the two-tiered approach will be even more difficult when patients have been previously vaccinated against Lyme borreliosis. Most ELISA or IFA screening tests use Bb that express large concentrations of OspA (25). Since vaccination induces seroreactivity against this protein, false- positive reactivities will become more frequent. As the number of OspA vac- cinees becomes larger, the cost of con- firmatory testing will also increase. Recently, Trevejo et al. (26) suggested WB confirmation only of serum samples yielding equivocal results in screening assays when patients were from areas highly endemic for Lyme borreliosis. The authors demonstrated superior test performance using this simplified pro- cedure and emphasized the resultant cost savings. However, Lyme.borreliosis vaccine recipients were not evaluated. Since vaccinees will react positively in screening assays and will most often reside in endemic areas, this approach will generate even more diagnostic uncertainty.

Confirmation by WB may also be more difficult after vaccination against Lyme borreliosis. Most WB procedures also use OspA-expressing Bb. Reactivi- ties against OspA proteins may obscure detection of other band reactivities, especially if OspA does not migrate as a single band. In addition, vaccinees will make antibodies to breakdown products of OspA that may make identification of low molecular weight proteins of Bb more diffUt by WB .

NOTE: No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise., or from my use or operation of any methods, products. instructions or ideas contained in the material herein. No suggested test or procedure should be carried out unless, in the reader’s judgment, its risk is justified. Because of rapid advances in the medical sciences, we recommend that the independent verification of diagnoses and drug doses should be made. Discussions, views and recommendations as to medical procedures, choice of drugs and drug dosages are the responsibility of the authors.

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Elimination of False Positives Due to Vaccination

Laboratories can eliminate cross- reactivity from screening assays by eliminating OspA from the detection antigen. Researchers have demonstrated that OspA-deficient Bb (27) or recom- binant protein antigens, including pri- marily OspC (28,29), can be used to detect early Lyme borreliosis with simi- lar sensitivities to currently used screen- ing assays containing OspA. However, these antigens are still highly nonspeci- fic. Thus, equivocal and positive reac- tions will still need confirmation by WB. Elimination of OspA reactivity from WB will be more difficult. To date, the ability of antigens from OspA- deficient mutants to detect Bb-specific antibodies has not been investigated.

Another option for confirming early Lyme borreliosis serodiagnostically is by detection of borreliacidal antibodies. We recently showed that viable Bb iso- late 50772, which lacks the genes ospA and ospB and expresses high amounts of OspC antigen (30,3 l), could be used to detect highly specific borreliacidal antibodies without decreasing sensitivity by using a flow cytometric borreliacidal antibody test (32). The enhanced speci- ficity is due to the detection only of antibodies that can specifically kill Bb. In contrast, bound cross-reactive anti- bodies are readily detected on non-viable Bb isolate 50772 when exposed to con- jugated anti-IgM or IgG in the ELISA or IFA. Since isolate 50772 is non-infec- tious, the flow cytometric borreliacidal antibody test can be performed safely.

We recently performed a CDC- sponsored blinded evaluation of the borreliacidal antibody test. Sixty serum samples consisting of 25 early Lyme borreliosis sera, 25 control sera, and ten split samples were tested. Each serum sample was also tested at the CDC by the two-tiered testing procedure. The borreliacidal antibody test and the two- tiered procedure detected early Lyme borreliosis with equal sensitivity (77%). These results were also identical to our earlier published results (30). Thus, the borreliacidal antibody test is as sensi- tive as the combined three tests used in the two-tiered method. In addition, the borreliacidal antibody test was highly specific. Including the CDC evaluation, we have tested 572 potentially cross- reactive sera including normal sera,

normal sera from patients vaccinated with the Lyme disease vaccine and sera from patients with potentially cross- reactive illnesses including Rocky Mountain spotted fever, assorted auto- immune diseases, tick-borne relapsing fever, Epstein-Barr virus, cytomegalo- virus, syphilis, and ehrlichiosis. Bor- reliacidal antibodies were detected in 16 (2.8%) of these samples and only small concentrations (120 titer) were detectable in 14 (87%). Since borreli- acidal antibody titers in early Lyme disease patients are detectable at high concentrations, a >99% specificity can be obtained with minimal effect on test sensitivity by adjusting the positive cut- off titer. This high specificity also will increase the accuracy of serodiagnosis, especially when testing sera from patients with a low pretest probability of Lyme borreliosis. In addition, the borreliacidal antibody test is not hin- dered by previous vaccination against Lyme borreliosis.

An Increasing Role for the Borreliacidal Antibody Test

An accurate diagnosis of Lyme bor- reliosis relies on both clinical and labo- ratory findings. However, serodiagnostic tests have been hindered by a lack of sensitivity and specificity. Furthermore, the lack of standardization of serologic tests has created difficulties in interpret- ing tests performed in different labora- tories. Collectively, these shortcomings have generated considerable confusion and have contributed to the increasing costs of medical care. The two-tiered testing system can perform well, espe- cially when serum is obtained from individuals with a high pretest proba- bility of Lyme borreliosis. If the pretest probability is low, however, the two- tiered approach results in more false- positive results. In addition, the widespread availability of the Lyme borreliosis vaccine renders most com- mercial screening tests obsolete which will make it necessary to detect Lyme borreliosis by subjectively evaluating Western blot results.

Detectable levels of highly-specific (>99%) borreliacidal antibodies are pro- duced shortly after infection with Lyme borreliosis spirochetes. these antibodies can be easily and objectively detected by flow cytometry. Antimicrobial agents, which could be expected to give false-

positive results, can be easily removed without interfering with the sensitivity of borreliacidal antibody detection (manuscript submitted, S.M Callister). The performance of the borreliacidal antibody test in a CDC evaluation, combined with the results from previ- ously published studies, validates the use of this single test for both screen- ing and confirming infection with B. burgdorferi. This sensitive and highly- specific single test is simple to perform, rapid, objective, and not affected by antibodies induced by the Lyme borre- liosis vaccine. Most importantly, the borreliacidal antibody test is less expen- sive than the two-tiered system. The inability to incorporate live spirochetes into a commercial kit could also improve Lyme disease serodiagnosis by confin- ing the borreliacidal antibody test to reference laboratories, especially state laboratories in endemic areas where it could be easily standardized.

The borreliacidal antibody test has been largely ignored, especially by the CDC. Research proposals designed to more completely define the utility of borreliacidal antibody detection (e.g., prognostic indicator of successful ther- apy) have been rejected and promises of more comprehensive collaborative studies have not been fulfilled. How- ever, there are currently more than 500 laboratories performing serodiagnostic tests for Lyme disease and serum from patients with a low probability of Lyme borreliosis are constantly being evaluated. The inaccurate results obtained by the two-tiered approach, especially in these patients, continues to cause confusion. Vaccination against Lyme borreliosis will further confound the accurate sero- diagnosis by this method. Widespread availability of a borreliacidal antibody test would greatly increase the accuracy of serodiagnosis of Lyme borreliosis. Perhaps the time is right to more com- pletely investigate this simple, altema- tive testing procedure.

Summary Approval of a Lyme borreliosis vac-

cine is an important first step. However, some individuals may still become infected with Bb despite vaccination. Therefore, vaccination will further con- found accurate serodiagnosis of early Lyme borreliosis by increasing the number of false-positive results in

Clinical Microbiology Newsletter 21: 16,1999 0 1999 Elsevier Science Inc. 0196-4399/99 (see fronhnatter) 131

screening tests commonly used in the two-tiered approach. In addition, the increased need for confirmatory testing will significantly increase costs and WB also may be affected adversely by seroreactivity against OspA. The borre- liacidal antibody test, which is not affected by vaccination, is a viable alternative to the two-tiered approach. The sensitivities of the two-tiered approach and the borreliacidal test are similar, however, the borreliacidal anti- body test offers greater specificity and is a single step test.

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