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.)
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
|
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
|
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.
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.
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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IGeneX, Inc.
797 San Antonio Rd., Palo Alto, CA USA 94303
Tel. 650.424.1191 / 800.832.3200 Fax. 650.424.1196
My thanks to Dr. Nick S. Harris, PhD, ABMLI for his helpful lessons and permission to post this article.