Journal of Spirochetal and Tick-Borne Diseases—Volume 2, Number 2; 1995

Detection of Borrelia burgdorferi Antigen
in Urine from Patients with Lyme Borreliosis

Nick S. Harris, PhD, and Boyd G Stephens, MD
IGeneX, Inc., Reference Laboratory, Palo Alto, California

Abstract: Lyme disease or Lyme borreliosis is a multisystem disease caused by infection with a spirochete Borrelia burgdorferi. In some patients diagnosed with Lyme borreliosis, the classical antibody response is slow or never develops. There are also reports of the antibody disappearing after antibiotic treatment. These reports and other enigma of Lyme disease often raise the clinical question of whether the reappearance of symptoms compatible with Lyme borreliosis after treatment are related to a reinfection or to the persistence of the original infection. The ability to observe antigenuria in Lyme borreliosis could aid in the clinical assessment and management of these difficult patients. This paper presents the development of an antigen assay for B. burgdorferi based upon detecting the presence of Borrelia antigen in the urine of patients with Lyme borreliosis and discusses the relationship of the antibody response to the presence of antigen. An antigen "capture" competitive inhibition assay was developed that can detect B. burgdorferi antigen in the urine of patients. Antigen was typically detected early in the course of disease, but it was also seen in some patients a year or more after the erythema migrans (EM) rash. In this method, antigen was captured by a unique polyclonal antibody before it could compete with antigen bound in the solid phase. The antibody used was a specifically absorbed polyclonal antibody, which had reactivity only against the 31, 34, 39, and 93 kDa antigens of B. burgdorferi. The affinity of the antibody and the nature of the assay allowed specific detection of low levels of antigen, in spite of the presence of other proteins. Serum and urine samples were obtained from more than 700 patients (425) and normal controls. After single-blind laboratory analysis, the results were correlated with clinical examination results and patient history. It was found that 30% of patients with Lyme disease (251 EM positive) had a positive Lyme Urine Antigen Test (LUAT) and 8% had a concurrent positive serology. The LUAT was positive in all three phases of disease: early (less than 60 days), before serology was positive; during treatment (60 days to 1 year); and a late period (greater than 360 days), when serology was often negative. Although Lyme borreliosis is defined by clinical diagnosis, various markers from the laboratory, such as specific antibodies and antigen, can aid in this process. The presence of specific antigen of B. burgdorferi in the urine of patients with Lyme borreliosis may be an adjunctive marker to the clinical diagnosis and traditional serological assays.

Key words: Lyme Disease, LUAT, Lyme Antigen Test, In-vitro assay, Antigen Capture, Urine

Lyme borreliosis is caused by an infection with the spirochete Borrelia burgdoferi, normally transmitted via a tick bite. This disease is reportedly the leading arthropod-borne illness in the United States and causes disease in Asia and Europe (1, 2).

Although Lyme borreliosis can respond favorably with early antibiotic treatment (3), the disease may be missed during its early stages (4). While erythema migrans (EM) is the characteristic diagnostic rash, it does not appear in all patients. Even in those patients in which EM appears, it may be overlooked or misdiagnosed as another type of insect bite. Clinical signs of the disease are frequently attributed to other causes. Symptoms can include a flu-like illness, headache, fatigue, muscle or abdominal pain, cardiac and neurological abnormalities, and arthritis (3, 5).

Current laboratory tests for Lyme borreliosis are serological assays to evaluate IgG and/or IgM specific antibodies to B. burgdorferi (4, 6, 7) and include IFA, enzyme-linked immunosorbent assay (ELISA), and Western Blot. However, there is no universally defined reference standard, and patients may test negative on one assay and positive on another due to immune response variability and the complex nature of the B. burgdorferi antigens (8, 9, 10). This could explain in part the reports of variability in results between laboratories (11). In addition, all serum antibody tests suffer from a higher false-negative rate in the early stages of the disease because antibodies may not be produced in detectable quantity until several weeks after infection (3, 6). It has also been suggested that early but inadequate antibiotic treatment may prevent full antibody development in clinically positive patients, mask clinical symptoms, and not completely eradicate the organism (6).

Testing accuracy or the ability to have greater sensitivity without sacrificing specificity increases in the later stages, but false-positives are still known to occur due to cross reactivity with syphilis, mononucleosis, some autoimmune diseases, and possibly periodontal disease (3, 9, 12). In addition, there is a report that in some cases, immune complexes may mask the serological response (13). Therefore, most clinicians recommend that a diagnosis of Lyme borreliosis be based on clinical signs and symptoms, with multiple laboratory tests being used only as supportive data (5, 8, 14). Assays that focus on the detection of some of the more unique antigens of B. burgdorferi may help provide additional laboratory tools to aid in the diagnosis of Lyme borreliosis.

In 1989, Hyde et al. (15), using multiple monoclonal antibodies in a dot blot assay, reported the detection of specific B. burgdorferi antigens (31, 34, and 41 kDa) in the urine of mice and humans. Coyle et al. (16) detected antigen in the cerebrospinal fluid (CSF) of neurological patients with presumed B. burgdorferi infection using monoclonal antibodies in an antigen-capture ELISA. Dorward et al. (17) used a rabbit polyclonal antibody in an electron microscopic, immune capture assay, and detected antigen of B. burgdorferi in the urine of mice and humans. Unique to this study was the observation that small fragments of Borrelia antigen, rather than whole organisms, were the more likely finding. Reports (18) have also indicated the detection of spirochetal DNA in the urine of patients with Lyme borreliosis. It was not clear from any of the above mentioned studies whether the bladder or the urinary tract itself was a unique site for the spirochete. A study of Magnarelli et al. (19) in mice detected infected bladders in 95% of mice with antigen in the urine.

The objective of the current report was to measure antigen in the urine and antibodies in the serum of patients with Lyme borreliosis. The Lyme Urine Antigen Assay (LUAT), thus developed, was a special antigen capture-inhibition ELISA with a unique absorbed polyclonal antibody with binding activity to 31, 34, 39, and 93 kDa antigenic moieties.

MATERIALS AND METHODS

Study design

The initial studies of the LUAT were designed to examine negative control groups as well as patients suspected of having Lyme borreliosis. The LUAT was performed single-blind on more than 700 patients and negative controls.

Serum and urine specimens from patients presenting with symptoms of Lyme borreliosis (n = 425) were submitted in a single-blind fashion to the laboratory. The minimal criteria (20) for inclusion in this group were tick bite, being from an endemic area, and three or more recognized symptoms of Lyme disease. Samples were sent frozen and kept at -20°C until analyzed. After analysis, a clinical study monitor assembled the data from a uniform history form that had recorded the data of a physician observed EM, other laboratory data, history, and current signs and symptoms. In addition, previous and current antibiotic treatments were noted.

After the initial data analysis, the clinical study monitor established a subgroup of patients (n = 251) meeting the tighter CDC surveillance case definition (21). These patients all came from a recognized endemic area of New Jersey, Connecticut, or New York: all had a physician-diagnosed EM; and all had three or more of the recognized clinical manifestations of Lyme borreliosis. Specimens were obtained from patients in all three phases of clinically diagnosed Lyme disease. The phases were defined as early, within 60 days of the EM; medium, between 60 days and 1 year of the EM; and late, more than 1 year after an EM.

The first normal control group (n = 208) was made up of individuals in an endemic area (Minnesota and Wisconsin, n = 139) and a non-endemic area (California, n = 69) with no symptoms or history of Lyme borreliosis or syphilis. A second control group (n = 50) came from the endemic area of New York and New Jersey. In addition, a third, special urine control group of patients with arthritic symptoms (n = 150) was established. All the patients, in the third control group, had either arthritis or arthralgias but no history or evidence of Lyme borreliosis, syphilis, systemic lupus (SLE), or scleroderma. This last group of patients came from all over the United States, with no geographic predominance.

Serological ELISA and antigen-capture ELISA

The FASTLyme serology assay was performed as previously reported (22). The overall format of the LUAT assay is presented in Figure 1. In the LUAT, antigen in urine competes with antigen bound on the solid phase. Captured antigen in the urine blocks the binding of the antibody to the solid phase and inhibits the development of fluorescence in the ELISA assay (23).

Figure 1.  Design of the LUAT

Negative controls were prepared from normal urine samples from healthy employees with no symptoms or indications of Lyme disease. Positive controls were prepared from assay positive urine samples from patients with clinically diagnosed Lyme borreliosis. Calibrators were made by spiking urine with sonicated B31 antigen at various concentrations. The 400 ng/mL calibrator was also used as one of the positive controls.

The LUAT assay was run in duplicate. The within-run co-efficient of variation (CV) was less than 10% and the run-to-run CV was less than 15%. One milliliter of thawed patient or control urines (pH 5 to 7) were spun at 12,000 X G for 10 minutes. Previous analysis showed that specific antigen of B. burgdorferi was found in both the pellet and supernatant but more consistently found in the pellet. Therefore, the supernatant was discarded. Since the pellet is used, urine specimens with gross cellular and gross bacterial contamination were excluded from the study. (We theorize that gross contamination may cause actual physical interference in the washing steps. From the blocking and spiking studies a reasonable amount of contamination has no effect on the assay system.) The pellet was resolubilized with 400 uL of 0.09 M Tris buffer at a pH of 7.4. This solution (pH 7.4 to 7.6) was then incubated with B. burgdorferi specific polyclonal antibody, conjugated with alkaline phosphatase for 1 hour at 37°C. Controls and calibrators were processed similarly.

The antibody used was from a unique pool of three rabbits hyperimmunized with sonicated, low passage, strain B31 of B. burgdorferi. To obtain the sonicate, 10 mL of a culture of B. burgdorferi were chilled in an ice bath and sonicated with a Tekmar sonicator (Cincinnati, OH), Tip Mod. No. CV 17 at a duty cycle of 60% and a tip limit of 3. Duration was for 1 minute, then pause for 1 minute. This cycle was continued until no whole spirochetes could be observed under the microscope with lOOX oil. The sonicated material was initially passed through a 0.8-um filter and then a 0.22-um filter. The rabbits were each initially injected with 500 mcg of sonicated B. burgdorferi antigen with Freund's complete adjuvant. They were boosted every 3 weeks with 100 mcg of antigen in Freund's incomplete adjuvant and test-bled until they had the appropriate response. This antibody was chosen because its reactivity to positive control samples most closely resembled three monoclonal antibodies developed by 3M Corporation (St. Paul, MN) against the Osp A (31 kDa), Osp B (34 kDa), and flagella (41 kDa) proteins and used in the initial published studies of a urine antigen test for B. burgdorferi (15). After absorption with common bacteria from both normal urines and from some patients with urinary tract infections (UTIs), Western blot analysis (Figure 2) of one of the strips cut from a run of negative and positive controls, patients and molecular weight markers demonstrated antibody activity only against 31, 34, 39, and 93 kDa antigenic moieties. The reactivity against 31 and 34 kDa appeared identical to that seen with the monoclonal antibodies (31 and 34 kDa) previously studied (15), and the reactivity to 39 kDa was distinct and different from that seen with the monoclonal antibody to 41 kDa.

Figure 2.  Western blot of the absorbed polyclonal antibody used in the LUAT

Microtitration wells (MicroFluor B, Dynatech Corp., Chantilly, VA) were pre-coated with the sonicated, low passage, B31 strain of B. burgdorferi. Wells were prewashed, and the test solution was added. Plates were incubated for 2 hours at controlled room temperature (21 to 23°C). The plates were then washed three times with the Tris buffered saline, pH 7.4, containing Triton X-405 detergent used to wash the pellet, and 100 uL of fluorescence substrate (4-methyl umbelliferyl phosphate) was added. The plate was then incubated for 20 to 30 minutes at controlled room temperature and read in a Dynatech fluorometer at 450 nm.

RESULTS

Blocking and interference studies

Blocking and interference studies were performed as follows: (1) negative patient urines were spiked with various concentrations of human serum protein; (2) negative patient urines were spiked with multiple concentrations of either whole blood, serum, RBCs, or WBCs; (3) antigen positive urines were spiked with either whole blood, serum, RBCs, or WBCs. Negative urines spiked as in action (1) or (2) above, remained negative, and no false-positives were detected. Antigen positive urines at values of 50 and 100 ng/mL retained 95 to 105% of their value when spiked with either HSA, blood, or blood components.

Normal control groups

An initial control group (n = 208) of individuals, characterized as negative for Lyme borreliosis by history and symptoms, was tested for the presence of antigen by the LUAT. These controls came from both an endemic area (Minnesota and Wisconsin, n = 139) and a non-endemic area (California, n = 69). This first control group had a 3% false-positive rate. Those seven control individuals who tested positive were lost to additional clinical follow-up.

A second control group (n = 50), more highly qualified than the first, was obtained (New York Biologics, Inc., New York, NY) from an endemic area of New York and New Jersey. All individuals in this group tested negative by the LUAT for the presence of any Lyme antigen.

Because of the high incidence of Lyme borreliosis patients appearing to have arthritic symptoms (Table 1), an additional control group was established. In this third study, urine from 50 patients from all over the United States, with arthritis and arthralgias, was examined. Patients were excluded from this study if they had Lyme borreliosis, syphilis, SLE, or scleroderma. Only one arthritic control exhibited a positive antigen value. Upon further study, this individual was found to have a UTI. This is the only listed contraindication to LUAT testing, because of the physical interference a large number of bacteria sometimes have with the ELISA format of the LUAT assay. (In the normal clinical practice of the laboratory all positive LUAT patients are tested by Multistix, Miles Inc. and any suspicion of UTI is reported back to the physician.)

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Table 1
Results of LUAT Testing in Total Population of Lyme Disease Patients (n = 425)

Characteristic	Number (n)	Percentage (%)
Physician-diagnosed EM	251	59
History of tick bite	210	49
History of both EM and tick bite	133	31
>3 other symptoms	380	89
History of arthritic symptoms	306	72
Positive concurrent serology	32	8
Positive Lyme Urine Antigen Test	124	29
Antibiotic treatment	261	61

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Table 2
Results of LUAT Testing in Lyme Disease Patients with a Physician- Diagnosed EM (n = 257)

Characteristic	Number (n)	Percentage (%)
History of tick bite	133	53
>3 other symptoms	204	81
History of arthritic symptoms	177	71
Positive concurrent serology	19	8
Positive Lyme Urine Antigen Test	75	30
Antibiotic treatment	159	63

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Lyme borreliosis patients

Patient samples from endemic areas of New York, New Jersey, and Connecticut were submitted to the laboratory. The samples were run single-blind by the laboratory. History and clinical information were stored and analyzed separately by the clinical study monitor. Of the patient samples submitted, only 425 patients met the criteria of a presumptive diagnosis of Lyme borreliosis and had a clinical history of either a physician-diagnosed EM or a tick bite with at least three major symptoms of Lyme borreliosis. The symptoms considered for the acute phase were "flulike" (which included fatigue, fever, headache, mild stiff neck, arthralgia, and/or myalgia). The symptoms considered for the later manifestations include any of the following when an alternate explanation is not found (20, 21): involvement of the musculoskeletal system, including arthritis in one or more joints; involvement of the nervous system, including lymphocytic meningitis, facial palsy, and radiculoneuropathy; and involvement of the cardiovascular system, including acute onset atrioventricular conduction defects. In all cases, concurrent serum and urine tests were performed on the samples.

The total data from this group of patients with Lyme borreliosis is presented in Table 1. Table 2 is a subgrouping of Table 1 and considers only those patients with a physician-diagnosed EM. The patients in Table 2 seem to meet the more stringent CDC Lyme borreliosis national surveillance case definition (21). It appears by an analysis of the data in both Tables 1 and 2 that the LUAT is positive in 30% of the patients. From a review of the patients' clinical history, more than 40% of the patients did have a positive serological response sometime in the course of their disease. However, only 8% of the patients in this current study were concurrently seropositive and antigen positive. The most common clinical finding in these Lyme borreliosis patients was the presence of arthritic symptoms.

Table 3 is an analysis, by phase of disease, of the patients with a physician-diagnosed EM who also had a positive LUAT. The arbitrary phases - early, middle, and late - were determined from the date of the EM defined by the diagnosing physician. From the analysis of these data, it appears that antigen can be detected both early and late in the disease process.

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Table 3
Analysis by Phases of Disease of Lyme Patients with EM and Positive LUAT (n = 75)^a

Early (<60 days)
>3 other symptoms	9/18	50%
Previous or current positive serology	2/18	11%
Medium (60 days to 1 year)
>3 other symptoms	21/24	88%
Previous or current positive serology	7/24	29%
Late (>1 year)
>3 other symptoms	27/29	93%
Previous or current positive serology	7/29	24%
Unknown
>3 other symptoms	4/4	100%
Previous or current positive serology	1/4	25%

  ^aStage of disease is defined from the time of the EM.

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DISCUSSION

These studies demonstrate that antigen of B. burgdorferi can be detected in the urine of a significant number of patients with Lyme borreliosis. Furthermore, there is a significant difference (p > 0.001) between the expression of this antigen in the urine of normal individuals from endemic and nonendemic areas as compared with patients with clinically diagnosed Lyme borreliosis.

There was no significant difference between control groups from endemic and nonendemic areas. Since arthritic symptoms were such prominent characteristics of patients with Lyme borreliosis, a comparison was made between non-Lyme patients with arthritic symptoms and the endemic and nonendemic normal controls. Again, there was no difference with respect to antigen detection between the normal controls and the patients with arthritis and arthralgias.

Those controls with the arthritic symptoms had less than a 1% false-positive rate, but that could reflect tighter entrance criteria used for this control study. There have been reports (24, 25) that some patients diagnosed with SLE or scleroderma may have DNA of B. burgdorferi in their blood or urine. The first control group studied did not exclude individuals with these diseases, but none were known to have been included. The arthritic symptom control study had specific exclusions for these conditions.

In the patient groups, a history of EM or tick bite with a combination of clinical symptoms were most effective in confirming Lyme borreliosis. However, the LUAT identified three to four times as many patients (124 versus 29) with Lyme borreliosis as the concurrent serology test (Table 1), possibly due to the pre-selection of patients, as previously described. Among patients with a history of EM (CDC surveillance criteria - Table 2), the LUAT identified 30%, while the antibody test identified only 8%.

Table 3 reviewed only the patients with EM and a positive LUAT, divided into phases of disease based on the initial appearance of the EM. This analysis suggests that antigen in urine is present at various times during all three phases of disease. The LUAT may be a useful diagnostic tool not only early in the disease process prior to the development of a serological response but also late in the disease process when the serological response has disappeared.

Some recent presentations (26, 27) have suggested the transient nature of antigen in urine. In those reports, antigen was present but not on a daily basis. This may be the explanation for the observation that the LUAT is sometimes negative in the face of an active infection. It was not clear from those studies whether the variation seen was due to assay performance or patient physiology. By use of the LUAT, those questions could be resolved because the LUAT is a highly controlled and reproducible assay. Future studies need to follow patients either daily or every other day after infection to monitor antigenuria. In addition, a weekly monitoring of serological response would be helpful.

There now exist a number of different tests for both antigen and antibody detection (12) for use with the clinical diagnosis of Lyme borreliosis. It is important to perform panels of tests in both serum and urine. This practice is done in other diseases, such as hepatitis, where multiple tests for both IgM and IgG antibody as well as tests for various types of antigens are routinely performed.

CONCLUSION

Lyme borreliosis is an increasingly common disease that is often difficult to accurately diagnose using only clinical symptoms. Without a physician-confirmed EM, it is particularly difficult to diagnose Lyme disease early in the disease process when treatment is most effective and long-term sequelae may be prevented. Recurrence of "Lyme-like" symptoms after antibiotic treatment is often a diagnostic dilemma. LUAT may prove to be a useful addition to current serological laboratory tests in assisting the differential diagnosis of Lyme borreliosis from other conditions presenting with similar symptoms. No single assay can work in all phases of diagnosis. Multiple laboratory tests should be used, with the clinical evaluation, to help in the diagnosis.

We would like to acknowledge the research staff of the former 3M Diagnostic Systems, Santa Clara, notably, John Scott, Geeta Kalbag, and Sunny Leung, and the clinical study monitors, Karen Meier and KC Yatsko. Special thanks to the medical technologists of 3M Diagnostic Systems, Estela Alabastro, Alana Hansen, and Norma Jovero, who ran the laboratory assays and are now with IGeneX, Inc. Reference Laboratory.

The above studies were originally approved by the IRB at 3M Corp., St. Paul, Minnesota.

Reprint requests: Nick S. Harris, PhD, IGeneX, Inc., Reference Laboratory, 797 San Antonio Road, Palo Alto, CA 94303.

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My thanks to Dr. Nick S. Harris, PhD, ABMLI for his helpful lessons and permission to post this article.