Spinning Lyme

"There have been some good reviews (4-6) prior to 1994 on the
laboratory aspects of diagnosis, but most of these were written before
the politicizing of the diagnostic process during the CDC/ASPHLD
meeting in Dearborn Michigan (7). "
http://www.igenex.com/labtest.htm
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Journal of Spirochetal and Tick-borne Diseases-Volume 5,
Spring/Summer 1998

An Understanding of Laboratory Testing for Lyme Disease
INTRODUCTION
While Lyme disease is a clinical diagnosis, the laboratory can provide
useful and necessary information for the diagnostic process. The
question is how does one use the laboratory in the most appropriate and
cost effective manner? The following examples stress the need for an
understanding of the tests available and when and why to use them.

HYPOTHETICAL CASE REPORT
A female patient in New Jersey presents to a university rheumatologist
with symptoms of arthralgia and myalgia, fatigue and malaise, rash,
photosensitivity, mild cognitive dysfunction, and nonspecific
gastrointestinal complaints. After a thorough physical examination, the
rheumatologist orders a WBC, multiple analyte chemistry panel,
sedimentation rate, ANA with a reflux to ENA if the ANA is positive,
rheumatoid factor, anticardiolipin, C3, C4, VDRL, urinalysis, and
perhaps some joint x-rays if the physical diagnosis is supportive.

This same patient also noted that one of her neighbors contracted Lyme
disease and believes she may have it as well. The rheumatologist then
added a screening test for Lyme disease, either ELISA or an IFA
quantitative (titer) test. If the ELISA or IFA were negative, the
chance of a diagnosis of Lyme disease in this patient would be remote
because the current dogma (1-3) is that Lyme disease is a rather rare
event in most parts of the country, especially in the absence of a
positive ELISA or IFA screening test.

BACKGROUND
There have been some good reviews (4-6) prior to 1994 on the laboratory
aspects of diagnosis, but most of these were written before the
politicizing of the diagnostic process during the CDC/ASPHLD meeting in
Dearborn Michigan (7). Prior to 1994, the CDC recognized Lyme disease
from a set of clinical symptoms and a general set of laboratory
findings. A certain combination of these criteria would lead to
diagnosis of Lyme disease that could be reported to the CDC. The
Dearborn meeting changed that.

The original clinical case definition (8) from the CDC for Public
Health Surveillance and reporting of Lyme disease was:

Clinical Criteria:

Erythema Migrans; or
At least one late manifestation of musculoskeletal, nervous or
cardiovascular system disorder; and laboratory confirmation.
Laboratory Criteria:

Isolation of Borrelia burgdorferi from clinical specimens; or
Demonstration of diagnostic levels of IgM and IgG antibodies to the
spirochete in serum or CSF (Western blot, ELISA, IFA), or
Significant changes in IgM or IgG antibody response to Borrelia
burgdorferi in paired acute- and convalescent-phase serum samples.
These criteria placed great emphasis on the presence of an Erythema
Migrans (EM) rash. It is usually accepted that a physician's diagnosis
of an EM on a patient from an endemic area is extremely useful for
diagnosing Lyme disease; almost a third of the patients actually do not
have an EM (9-11). In addition, the variability of the EM rash (12,13),
such as its duration, nonpruritic and nonpainful nature, and its
location in obscure areas (axilla and hair regions) inhibit its use as
a consistent diagnostic marker.

In 1995, the CDC added the additional recommendation from the
CDC/ASPHLD meeting (7) of a two-tiered approach for reporting active
disease and previous infection. That requirement means that a positive
sensitive ELISA/IFA must be followed by a positive Western blot with a
defined number of approved antibody bands. If the intention were only
for public health surveillance and reporting of disease, these changes
would not have caused a problem. Unfortunately, these recommendations
became the standard in most areas and especially with insurance
companies. That was unfortunate because the Dearborn meeting was not
supposed to be about setting national standards for Lyme disease
diagnosis; rather, it was to be a discussion regarding the Western blot
during early Lyme disease. The majority of patient samples used to set
the criteria were from patients being followed for four months
following their diagnoses. The patients considered for entry in the
study had an EM rash and either arthritis or neuroborreliosis.

Lyme disease is a problematic diagnosis because it is a complicated
clinical entity. The position by the CDC makes it more complex. Some
patients do not elicit an antibody response great enough to be positive
by the currently available ELISA assays. Recent studies (14) by the
group responsible for Lyme disease proficiency testing for the College
of American Pathologists (CAP) came to the conclusion that the
currently available ELISA assays for Lyme disease do not have adequate
sensitivity to meet the two-tiered approach recommended by the
CDC/ASPHLD group (7). In addition, Bakken et al (14) stated that a
screening test must have sensitivity >95% to adequately screen for Lyme
disease and that the currently available ELISA tests do not meet this
criteria. Furthermore, if patients are treated early with antibiotics,
their antibody response may be reduced or curtailed (15). The initial
mild flu-like symptoms may be overlooked. Later, if the symptoms
return, most of the antibody markers have disappeared. The picture is
not entirely bleak if Lyme disease is approached for what it is: a
complicated clinical entity, which requires multiple laboratory tests
to assist in the diagnosis. Thus, if clinicians use multiple tests (ie,
both screening and confirmatory Western blot assays, antigen-capture
and PCR), as they do in other disease entities, there will be fewer
problems with the diagnosis and fewer patients will be missed.

The presence of detectable spirochetes in infected tissue is rare. The
characteristic sparsity of organisms contributes to the difficulty of
getting blood or tissue to grow the Lyme bacterium (15). A positive
culture may not be a predictor of an antibody response. Rawlings (16)
followed a group of 14 patients in which she was able to culture B.
burgdorferi, but only 3 of those patients had positive antibody titers.
Aguero-Rosenfeld et al (12,13) showed that only 70% of the documented
Lyme patients in their study had a significant antibody response. They
suggested that the degree of antibody response might be related to the
length of time the EM rash persists. They also saw only a 64% rate of
IgM to IgG seroconversion.

Early reports suggested that considerable interlaboratory and
intralaboratory variability exist in Lyme disease testing (17-19).
However, a review of the 1996 Lyme proficiency results by CAP (College
of American Pathologists) and those by New York State demonstrates
comparable agreement between the laboratories, similar to other
bacterial infections and autoimmune conditions.


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Table 1. Assays for Lyme disease

Direct
Biopsy
Culture
Antibody Assays
IFA
ELISA
Western blot
Antigen Assays
Antigen-Capture
PCR


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RESULTS AND DISCUSSION
Table 1 presents the types of tests that are most commonly available
for Lyme disease. To provide adequate support for the clinical
evaluation, multiple tests should be used. Not only is a correct
diagnosis advantageous for the patient, but also ultimately is the most
cost effective.

Indirect fluorescent antibody (IFA)
B. burgdorferi spirochetes are affixed to glass slides and usually a
fluorescent-conjugated goat antihuman immunoglobulin of either IgM or
IgG specificity is used (20). Tests for Lyme disease using IFA have
received mixed reviews and some authors believe that the
interpretations of IFA assays are overly subjective and that the tests
are either functionally insensitive for Lyme-specific antibodies or
display considerable cross-reactions with antibodies to other
spirochetal organisms (21,22). Magnarelli et al (23,24) and Mitchell et
al (20) supported IFA if used in conjunction with a clinical
evaluation. Mitchell's study with the IgM IFA showed excellent
specificity and no observed cross-reactivity with infectious
mononucleosis (n = 20), rheumatoid arthritis (n = 19), systemic lupus
(n = 22), syphilis (n = 13), streptococcal sequelae (n = 20) or healthy
subjects. Mitchell related the success of this test to the quality of
the substrate slides and the level of experience of the technologists,
and concludes that IFA microscopy becomes less subjective with
experience.

Enzyme-linked immunosorbant assay
ELISA for B. burgdorferi has been available since 1984 (25). Most
commercial assays use a whole cell sonicate of B. burgdorferi. Complete
descriptions of methods for a Lyme ELISA can be found in the
publications by Craft et al (25), Magnarelli et al (23), and Russell et
al (21). Standard ELISA techniques have been employed (26) in all these
assays.

There are a large number of commercial ELISA tests available. A review
of past proficiency events by CAP and the NYS Health Department show
the relationship between the various tests. Most commercial ELISA tests
have comparable sensitivity and specificity because they were made to
compare to one another for the FDA 510K process. However, most are
inadequate as a screening test because they were not designed by the
manufacturers to be sensitive at the 95% level, which is required for
screening (14). A substantial change in the 510K approval process would
be required to make the ELISAs for Lyme disease diagnosis more
sensitive.

The goal for a new generation of ELISAs should be sensitivity for the
more unique and specific B. burgdorferi antigens that are visualized in
the Western blot (Figure 1). They are Osp A (31 kDa), Osp B (34 kDa),
Osp C (23-25 kDa), 39 kDa, and 93 kDa (27-32). Initially, some
investigators identified 93 kDa as 94 kDa and Osp C as 22 kDa. While
most ELISAs do have reactivity to these antigens, because they are
prepared with a sonicate of B. burgdorferi, they also have reactivity
against 41 kDa, 58 kDa, 66 kDa, and 73 kDa. While the later antigens
are components of B. burgdorferi, they also have considerable
cross-reactivity to other spirochetes, heat-shock proteins, and some
viruses (33).

All borderline and positive ELISA assays (polyvalent, IgG only, and IgM
only) for Lyme disease must be confirmed by a high quality Western blot
for B. burgdorferi. A 56% false-negative rate, depending upon the
commercial kit, was found by Luger and Krause (18), as compared to
their own clinical diagnoses. Golightly et al (34) saw a lack of
sensitivity with a 70% false-negative rate in early Lyme disease and
from 4% to 46% with late manifestations of Lyme disease. These results
support the necessity of Western blot confirmation for both positive
and negative Lyme ELISA.

B. burgdorferi Western blotting


Figure 1. Western blots of B. burgdorferi B31 strain: The blots were:
(1) stained with amino black, (2) reacted with rabbit antisera, (3)
with goat antisera, (4-7) with various monoclonal antibodies, and (8)
with pooled patient sera. Reproduced by permission from Ma B, Christen
B, Leung D, Vigo-Pelfrey C. Serodiagnosis of Lyme borreliosis by
Western immunoblot: reactivity of various significant antibodies
against B. burgdorferi. J Clin Microbiol 1992; 30: 370-76.

The immunoblot or Western blot (Figure 1) for B. burgdorferi is the
most useful antibody test available when performed in a quality
laboratory by experienced testing personnel. It is necessary to
evaluate separately both the IgM and IgG antibodies of B. burgdorferi.
The study by Ma et al (35) gives an excellent overview of the technique
and provides comprehensive information about the antibodies seen in
Lyme disease patients versus the normal and non-Lyme disease groups.



Figure 2. Western blots to B. burgdorferi from various patients with
Lyme disease Lanes 1-48 are IgG/IgM blots from clinically confirmed
patients with various levels of antibodies. Lanes 48-57 are IgM-only
blots. Reproduced by permission from Ma B, Christen B, Leung D,
Vigo-Pelfrey C. Serodiagnosis of Lyme borreliosis by Western immunoblot
reactivity of various significant antibodies against B. burgdorferi. J
Clin Microbial 1992; 30: 370-376.

Figure 2 illustrates a group of IgG-IgM Western blots (lanes 1-48) from
clinically confirmed Lyme patients with various levels of antibodies to
B. burgdorferi. In this figure are IgM Western blots to B. burgdorferi
(lanes 48-57). While some of these patients have different patterns of
antibody reactivity, all were confirmed, clinically positive Lyme
patients with physician-diagnosed EM. The variability in the Western
blot is characteristic of the variability observed in the immune
response of other diseases (eg, Hashimoto's thyroiditis, SLE, Sjogren's
syndrome, scleroderma). Our own clinical study of 186 defined patients
and 320 negative controls (Figure 3) demonstrated excellent sensitivity
and specificity for IgM using any two of the following bands: 23-25 kDa
(Osp C), 31 kDa (Osp A), 34 kDa (Osp B), 39 kDa and 41 kDa (35). This
study also demonstrated good specificity and sensitivity for IgG using
any two of the above bands. The 83/93 kDa antibody could also be
included as one of two IgG bands.



Figure 3. Comparison of antibody reactivity to various B. burgdorferi
antigens. The dark bars are from 186 patients with clinically confirmed
Lyme disease and the light bars are from 320 normal controls.
Reproduced by permission from Ma B, Christen B, Leung D, Vigo-Pelfrey
C. Serodiagnosis of Lyme borreliosis by Western immunoblot: reactivity
of various significant antibodies against B. burgdorferi. J Clin
Microbial 1992; 30: 370-76.

It is difficult for each laboratory to perform clinical studies and
establish its own ranges for normal and disease populations. For this
reason, the CDC assembled a group of academic scientists with the
assistance of the FDA and the Association of State and Territorial
Public Health Laboratory Directors (ASTPHLD) to reach a consensus on
certain criteria for the Western blot. After several meetings they
arrived at the CDC/ASPHLD consensus criteria presented in Table 2
(7,36). These criteria were based in large part on the work of Dressler
et al (37), using well-defined patients with active Lyme arthritis or
neuroborreliosis. Interestingly, in their publication none of the three
CDC/ASPHLD recommended strains of B. burgdorferi (B31, 297 and 2591)
were used. Rather, they used G39/40 with a 10% acrylamide gel, although
a gel with less than 11% of acrylamide does not have enough resolution
nor definition of all the important antigens of B. burgdorferi.

The criteria for a positive Western blot to B. burgdorferi developed by
the CDC/ASPHLD are very conservative and require 5 of 10 antibody bands
for IgG positivity; the original recommendations do not even recognize
equivocal or borderline results if less than five bands are detected.
Their cut-off assumes that all Lyme patients have similar immune
systems. They ignore the diversity of the immune response seen in other
diseases. Their studies were problematic in that they primarily focused
on patients with early (usually within four months of an EM) Lyme
disease. They drew blood in most patients every two weeks during this
four-month period and any positive event (five out of ten bands) was
counted as a positive patient, even if they were negative at a
different time of the study. In addition, the criteria include
antibodies to 41 kDa, a common antigen of most flagella-bearing
organisms, and exclude two of the most important and specific antigens,
31 kDa (Osp A) and 34 kDa (Osp B), which appear later in the response.
A review by Hilton et al (38) in a group of 50 patients with confirmed
Lyme disease showed that they would have missed 4 patients by excluding
31 kDa (Osp A) and 34 kDa (Osp B). The author's own laboratory would
have missed 2 of 18 proficiency samples by excluding antibodies to
these two antigens.

Engstrom et al (11) and Aguero-Rosenfeld et al (12,13) confirmed that
almost one-third of all Lyme patients are IgG seronegative during the
first year. Two years after physician-diagnosed EMs, 45% of the
patients were negative by ELISA. In another study, Aguero-Rosenfeld et
al (13) showed that the ELISA response declined much more rapidly than
the Western blot response. Their study also demonstrated that the
two-step protocol of the CDC/ASPHLD criteria would fail to confirm
infection in some patients with culture-proven EM. Furthermore,
although a majority (89%) of patients with EM rash developed IgG
antibodies detected by Western blot sometime during disease, only 22%
were positive by the criteria of the CDC/ASPHLD (13). The Engstrom et
al study (11) did not use the IgG blot criteria of the CDC/ASPHLD. They
found that 2 of 5 bands gave them a specificity of 93 to 96% and a
sensitivity of 100% in the 70% of patients who made antibody. This
might imply that they would have had even less sensitivity had they
used the more stringent CDC/ASPHLD criteria.

The CDC/ASPHLD criteria (7,36) for a positive IgM Western blot include
the 23-25 kDa (Osp C), the 39 kDa, and the 41 kDa, but exclude the 31
kDa (Osp A) and 34 kDa (Osp B). During the presentation at the Dearborn
meeting (7), the specificity of the IgM Western blot was reported to be
greater than 95% based on several hundred negative controls. Engstrom
et al (11) reported specificities of their IgM Western blot to be
between 92% and 94%. It has been reported that the IFA and ELISA IgM
assays may show cross-reactivity with ANA, EBV, and spirochetal
infections (24). However, studies by Mitchell et al (20) and Ma et al
(35) did not observe this with their IFA and Western blot assays
respectively.

A major disagreement with the CDC/ASPHLD group is with its statement
that the IgM Western blot should only be used during the first month
after tick bite. They have overlooked their own reported excellent
specificity of the IgM Western blot. The author's laboratory (35),
studies by Steere (28), and by Jam et al (40) point to the importance
of the IgM Western blot in recurrent and/or persistent disease. Sivak
et al (41) found that the IgM Western blot had a 96% specificity if the
patients had at least a 50% probability of having Lyme disease. A study
by Oksi et al (42), using culture and PCR to confirm Lyme disease,
reported that specific IgM to B. burgdorferi is sometimes the only
antibody detected in persistent disease. They felt that this data
supported the idea that some Lyme patients have a restricted IgM-only
response to B. burgdorferi Lyme disease.

It is important to note that a positive IgG and/or IgM Western blot
only implies exposure to B. burgdorferi. It is only part of the test
battery and is not confirmatory for Lyme disease. It does not mean the
patient has Lyme disease; that is a clinical diagnosis. It must also be
kept in mind that these antibody tests are not static; they change over
time. A patient negative in the Western blot may seroconvert to a
positive pattern with treatment. Conversely, a patient could redevelop
an IgM response, suggestive of a recurrent infection.


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Table 2. CDC/ASPHLD criteria for the serologic diagnosis of Lyme
disease

Test Performance and Interpretation

Recommendation 1.1. Two-Test Protocol
All serum specimens submitted for Lyme disease testing should be
evaluated in a two-step process, in which the first step is a sensitive
serological test, such as an enzyme immunoassay (EIA) or
immunofluorescent assay (IFA). All specimens found to be positive or
equivocal by a sensitive EIA or IFA should be tested by a standardized
Western blot (WB) procedure. Specimens found to be negative by a
sensitive EIA or IFA need not be tested further.

Recommendation 1.2. WB Controls
Immunoblotting should be performed using a negative control, a weakly
reactive positive control, and a high-titered positive control. The
weakly reactive positive control should be used to judge whether a
sample band has sufficient intensity to be scored. Monoclonal or
polyclonal antibodies to antigens of diagnostic importance should be
used to calibrate the blots.

Recommendation 1.3. Testing and Stage of Disease
When Western immunoblot is used in the first four weeks after disease
onset (early Lyme disease), both IgM and IgG procedures should be
performed. Most Lyme disease patients will seroconvert within this
four-week period. In the event that a patient with suspected early Lyme
disease has a negative serology, serologic evidence of infection is
best obtained by the testing of paired acute- and convalescent-phase
samples. In late Lyme disease, the predominant antibody response is
usually IgG. It is highly unusual that a patient with active Lyme
disease has only an IgM response to Borrelia burgdorferi after one
month of infection. A positive IgM result alone is not recommended for
use in determining active disease in persons with illness of longer
than one month duration, because the likelihood of a false-positive
tests result is high for these individuals.

Recommendation 1.4. WB Criteria
Use of the criteria of Engstrom et al (11) are recommended for
interpretation of IgM immunoblots. An IgM blot is considered positive
if two of the following three bands are present: 24 kDa (Osp C), 39 kDa
(Bmp A), and 41 kDa (Fla).
Monoclonal antibodies to these three proteins have been developed and
are suitable for calibrating immunoblots (7).
Once antibodies are developed to the 37 kDa antigen, this protein could
be used as an additional band for IgM criteria (>2 of 4 bands).
Interim use of the criteria of Dressler et al (37) are recommended for
interpretation of IgG immunoblots. An IgG blot is considered positive
if five of the following ten bands are present: 18, 21 (Osp C), 28, 30,
39 (Bmp A), 41 (Fla), 45, 58 (not GroEL2), 66 and 93 kDa. Monoclonal
antibodies have been developed to the Osp C, 39 (Bmp A), 41 (Fla), 66,
and 93 kDa antigens and are suitable for calibrating IgG immunoblots
(7).
The apparent molecular mass of Osp C is recorded above as it was
denoted in the published literature. The protein referred to as 24 kDa
or 21 kDa is the same, and should be identified in immunoblots with an
appropriate calibration reagent (see 1.6).

Recommendation 1.5. Reporting of Results
An equivocal or positive EIA or IFA result followed by a negative
immunoblot result should be reported as negative. An equivocal or
positive EIA or IFA result followed by a positive immunoblot result
should be reported as positive.
An explanation and interpretation of test results should accompany all
reports.

Recommendation 1.6. Standardization of WB Nomenclature
The apparent molecular mass of some proteins of Borrelia burgdorferi
such as Osp C will vary depending on the B. burgdorferi strain and gel
electrophoresis system used. The molecular weights of proteins of
diagnostic importance should be identified with monoclonal or
polyclonal antibodies. When possible, the molecular weight of the
protein should be followed by the descriptive name (eg, Osp C).

MMWR 1995; 44: 590-91


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Antigen and antigen-capture assays for Lyme disease
Several studies, using mice, rats, guinea pigs, and dogs have found B.
burgdorferi antigen in the urine of naturally occurring and
experimentally induced Lyme infections (43-46). Dorward et al (44) and
others (43,47) also detected antigen in the urine of patients with Lyme
disease. Dorward's study (44) indicated that pieces or blebs of B.
burgdorferi were more commonly found in urine than was the entire
organism. Coyle et al (48) has successfully used antigen-capture with
monoclonal antibodies to 31 kDa (Osp A) and 34 kDa (Osp B) to detect
antigen in the cerebrospinal fluid (CSF) of patients with
neuroborreliosis.

Harris and Stephens (50) have presented information about the
development and use of antigen-capture for the detection of B.
burgdorferi antigen in the urine of Lyme patients. The antibody used in
this antigen-capture is a unique polyclonal antibody that is specific
for the 31 kDa (Osp A), 34 kDa (Osp B), 39 kDa, and 93 kDa antigens of
B. burgdorferi. The assay appears to be very specific for these
antigens of B. burgdorferi, and in 408 controls there was less than a
1% false positive rate. Furthermore, blocking and interference studies
with human RBCs, WBCs, whole blood, serum and human serum albumin
showed no effect on the urine or CSF antigen-capture assay (50).

Urine and serum from 251 patients with Lyme disease (confirmed after a
physician-diagnosed EM rash) were studied for the concurrence of a
positive ELISA and a positive antigen test. In Table 3 it can be seen
that 30% of this group of Lyme disease patients had a positive Lyme
Urine Antigen Test (LUAT), but a concurrent positive IgG/IgM ELISA was
only seen 8% of the time. Other studies (51) have suggested that
antigenuria may not be a constant daily occurrence. Therefore, multiple
sampling days for urine may be more effective for detecting antigenuria
than a single collection (39).


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Table 3. Patients with physician diagnosed EM n = 251


History of tick bite 33/251 53%
>3 other symptoms 204/251 81%
History of arthritis 177/251 71%
Positive concurrent ELISA 19/251 8%
Positive LUAT 75/251 30%


Harris NS, Stephens BG. Detection of Borrelia burgdorferi antigen in
urine from patients with Lyme borreliosis. J Spirochet Tick-Borne Dis
1995; 2: 37-41.


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Polymerase chain reaction (PCR)
The PCR assay for B. burgdorferi looks for the presence of B.
burgdorferi DNA commonly in blood, CSF, urine, and synovial fluid.
There are many published articles that provide background (52-56) to
this topic.

As mentioned previously, Lyme disease is characterized by a sparsity of
organisms (15). Some laboratories perform the genomic assay, which
requires a minimum of one recoverable bacterium, or at least the DNA
from one. A plasmid PCR assay is also available from some laboratories.
Dorward et al (44) using an immune electron microscopic technique,
detected pieces of antigen rather than intact organisms in urine and
other tissues. In an earlier study, Garon et al (57) detected blebs or
membrane vesicles shed from the surface of B. burgdorferi. These blebs
contain the same antigen as the intact organism (Dorward, personal
communication). These blebs and fragments of B. burgdorferi antigen may
be the reason that the antigen capture and plasmid PCR demonstrate
great practical sensitivity. Nocton et al (54) reported on the use of a
plasmid PCR that had excellent sensitivity in the synovial fluid of
patients with Lyme arthritis.

Studies by Goodman et al (55) found that 30% of their patients with
early Lyme disease were positive by PCR. This is comparable to blood
culture data by other groups (55). However, some groups cannot find
positive cultures or positive PCR from patients with acute Lyme disease
(59). This is definitely an area that is technique dependent. Manak et
al (60) was able to detect 33% of early Lyme patients and 50% of late
stage Lyme disease in patients not on antibiotic therapy. Most patients
become PCR negative within two weeks of antibiotic therapy. They also
saw that during a relapse, patients might become PCR positive for a
short period of time.

Schmidt et al (56) found that urine samples from 22 of 24 patients with
an untreated EM rash were positive using a nested PCR for B.
burgdorferi sensu stricto as well as reactive to B. garninii and B.
afzelii but not to B. hermsii. Immediately after the initiation of
therapy (minocycline, 100 mg BID for 14 days) 58% were still PCR
positive. Twenty weeks after therapy, none of the patients were
positive. Bayer et al (61) on the other hand, using a combination of
genomic and plasmid PCR on urine samples, found that 74% of patients
with chronic (persistent) Lyme disease were PCR positive. These
patients had been treated between three weeks and two months
continually with antibiotics, but were off antibiotics one week prior
to the test.



Figure 4. A model suggesting the tests applicable in different phases
of Lyme disease. The left side of the figure indicates the hypothetical
patient making antibodies after infection. Less than 70% of patients
are in this category. The responses shown on the right side of Figure 4
pertain to many of the patients with recurrent/persistent disease.
Courtesy of IGeneX, Inc. Reference Laboratory, Palo Alto, CA.

Which tests to use?
The physician should have a logical plan for choosing the laboratory
tests to be used initially, and what type of follow-up tests to order
if additional information is needed to aid in the diagnosis of Lyme
disease. Similar to the hepatitis model, B. burgdorferi antigen is
present early after infection. B. burgdorferi DNA in urine has been
detected by PCR within the first few weeks after infection (56).
Studies by the author using LUAT have found antigen as early as three
days after a tick bite (unpublished observations).

Within two to four weeks after infection, an IgM antibody response to
B. burgdorferi may be detected in 60 - 70% of the patients. This is
followed by a specific IgG response, which may remain detectable for a
few months or in some cases, a few years. In the early period,
especially during the EM, it may also be possible to detect by PCR B.
burgdorferi DNA in the urine and/or blood (55,56).

To evaluate a new patient at any stage of disease, at least an IgM and
an IgG Western blot must be performed. For completeness, an ELISA or
IFA screening test may also be ordered. Contrary to popular thought
(1,2), most ELISAs and IFAs do not have enough sensitivity to be used
as a screening test (14). The Western blot is more sensitive and
specific. The increased sensitivity of the Western blot is analogous to
a mountain where the base is a Western blot and the summit is an ELISA.
The Western blot has considerably more sensitivity because it provides
detection before the peak of the response. As mentioned before, the
Western blot is a qualitative assay based upon a visualization of a
patient's antibody against the various unique B. burgdorferi antigens.
This type of assay should not be restricted by the same sensitivity and
specificity concerns as a general screening test. An ELISA with a
quantitative or semi-quantitative cut-off is usually not specific to
only the unique B. burgdorferi antigens. However, an ELISA assay
developed to cloned antigens of B. burgdorferi, would most likely have
more analytical sensitivity than the Western blot.

Some of these relationships may be seen in the hypothetical model of an
"idealized" B. burgdorferi infection (Figure 4). The left side of the
figure may be valid early in disease in the two-thirds of patients
making antibody. In the other third of patients, or later than the
first year or with persistent/recurrent disease, the right side of
Figure 4 may pertain. Therefore, a specific battery of tests (as used
with other diseases such as hepatitis, thyroid dysfunction, or
autoimmunity) provides a more complete picture to help with the
clinical diagnosis and is ultimately more economical for the patient.

Persistent/recurrent (chronic) infection is a unique diagnostic problem
because the IgG response may be absent in more than 50% of the patients
(11-13). Thus, in addition to the IgG Western blot, an IgM Western blot
should be used. This technique has been helpful for some patients with
persistent/recurrent disease (28,42). The physician must rule out
possible cross-reactions from rheumatoid factor, other spirochetal and
tick-borne diseases, and infectious mononucleosis (18,24). This can
usually be accomplished during the differential diagnostic process. In
addition, a recent study has indicated that the IgM Western blot may be
as high as 96% specific, with almost a 93% predictive value of disease,
if the patient has at least a 50% prior probability of Lyme disease
(41).

Assays that focus on antigen detection or DNA may be particularly
useful diagnostically during persistent/recurrent disease (50). Antigen
capture in urine has been a useful diagnostic tool, especially during
the initiation of new antibiotics, which seems to enhance antigenuria
(39). However, antigen capture assays in urine (LUAT) should only be
used after patients have been properly evaluated by sensitive antibody
assays. Studies have shown that patients seropositive to B. burgdorferi
have less antigenuria than seronegative patients (50).

The PCR and the Lyme Urine Antigen Test (LUAT) are sometimes
complementary. As mentioned, patients responding to antibiotics may
have a negative PCR. While a genomic PCR requires one recoverable
bacterium or at least the DNA from one, studies at the Rocky Mountain
National Laboratory have shown that pieces of antigen are more commonly
found in urine than are whole or semi-whole B. burgdorferi (44). In
addition to the Western blot, PCR and antigen capture can be used for
testing the synovial fluid of inflamed joints, a common occurrence in
Lyme disease. The plasmid PCR for B. burgdorferi in synovial fluid was
used as a diagnostic aid for patients with Lyme arthritis (54). This
study showed that 96% of the patients with untreated Lyme disease and
those treated with only a short course of antibiotics had a positive
PCR assay of their synovial fluid.

Tests for neurological Lyme disease
A wide range of neurological symptoms has been reported in Lyme
disease. They include Bell's palsy, meningitis, meningoencephalitis,
radiculoneuritis, encephalopathy, psychiatric syndromes, fatigue,
multiple sclerosis-like symptoms, and Parkinson-like symptoms
(48,62-69).

Diagnostic assays for neurological Lyme disease must evaluate the CSF
(70-72). According to Coyle, the blood of the brain is CSF, and it is
impossible to make a diagnosis of neurological Lyme disease without
performing a spinal tap and analyzing the CSF for antibodies and
antigens to B. burgdorferi (personal communication). One assay that has
been commonly used is the CSF to serum index; it is a combination of
immunological tests that measures specific antibodies to B. burgdorferi
in both serum and spinal fluid. Calculations are based on the results
of quantitation of IgG in both the serum and CSF, as well as on the
results of the CSF and serum ELISA. An index greater than one (>1.0) of
the CSF:serum ELISA suggests in situ synthesis of antibody in the
central nervous system. The use of an index is important because if a
test were only performed on the CSF, there would be no control for
leakage across the blood-brain barrier. Unfortunately, this series of
tests uses the same flawed ELISA assays as those used on serum.
Therefore, sensitivity is a concern. A positive result is serological
evidence of neuroborreliosis, whereas a negative result indicates only
that antibody was not detected, not the absence of disease.

Because of sensitivity concerns reported with the ELISA, the IgM and
IgG Western blot is the antibody test of choice for the CSF, but the
two tests require 2 mL of CSF. A positive result with either the IgG or
IgM Western blot is serological support of neurological Lyme disease. A
recent study (73) confirmed what has been observed for some time in the
author's laboratory, that is: specific B. burgdorferi proteins such as
Osp A and Osp C may also be seen in the CSF in early neurological Lyme
disease using an IgM Western blot. Since it is always necessary to
control for leakage across the blood-brain barrier, CSF Western blots
should be compared to those on the patient's serum. Tests in the
author's laboratory suggest that the detectable level of antibody,
using standard techniques with the Western blot, is 50 - 100 ng/mL of
specific antibody. This would imply that for practical purposes the CSF
should contain at least 1 ug/mL of immunoglobulin before doing an
assay.

PCR and antigen capture assays using CSF have been useful in some
patients with neurological Lyme (47,48,74), especially since some
patients with neurological Lyme disease are negative for Borrelia
antibody in the CSF (63,68,74). These patients may also be negative for
all assays in blood and urine. A recent study by Fallon et al (75)
suggested that brain imaging using a single photon emission computed
tomographic (SPECT) technique is another diagnostic approach for
neurological Lyme disease.

Tests for associated tick-borne diseases
There appears to be an association between Lyme disease, and
ehrlichiosis and babesiosis (76-81) and the same type of tests (IFA,
ELISA, Western blot and PCR) used for Lyme disease (Table 1) can be
used for these associated tick-borne diseases. Usually, however, the
IFA test is more commonly available.

Human ehrlichiosis is a disease caused by rickettsial-type organisms
transmitted by some of the same ticks that carry Lyme disease. Human
Granulocytic Ehlichiosis (HGE) has been closely linked to the bites of
Ixodes scapularis and Ixodes pacificus (82,83). Human Monocytic
Ehrlichiosis has been linked to the bites of Amblyomma americanum (Lone
Star tick) (82,83). Currently, IFA serology is performed using E.
chaffeensis (84) for HME, and the closely related E. equii (85) or the
newly discovered organism for HGE (86).

Ehrlichiosis usually presents with high fever, malaise, headache,
myalgia, sweats, and nausea. These patients generally have high titers
(>1:1000) during or shortly after this acute disease. Those patients
diagnosed with Ehrlichia should also be tested for Lyme disease, since
the same tick transmits the disease and co-infections have been noted
(79,81).

Babesiosis is another disease transmitted by the same tick that carries
B. burgdorferi (76-80). Symptoms of babesiosis are also similar to some
of the symptoms of Lyme disease: fatigue, malaise, myalgia, arthralgia,
chills and fever. Usually the fever is high. This disease is
particularly life threatening in splenectomized or immune suppressed
patients.

Babesiosis is caused by an intraerythrocytic parasite, Babesia microti
(78,87), which is similar to Plasmodium falciparum, the causative agent
of malaria. In fact, many of the symptoms and the appearance of ring
shaped intraerythrocytic parasites in red cells stained with Giemsa or
Wright's often leads to the incorrect diagnosis of malaria. Serology by
IFA is done using red cells from infected Syrian hamsters. The antibody
titers are usually high (>1:640) in acute babesiosis, and the piroplasm
can be seen in the red blood cells of patients. Seroconversion usually
occurs between two and four weeks after infection.

Lower levels of antibody to B. microti, E. chaffeensis, and E. equii
have been seen in some patients diagnosed with Lyme disease. The
significance of these antibodies is not understood and it is not known
if they represent a subclinical infection of babesiosis (95) or
ehrlichiosis associated with Lyme disease, or if they are merely low
levels of insignificant antibody.

CONCLUSION
Antibody assays for Lyme disease will improve when recombinant antigens
become available to the unique antigens of B. burgdorferi (30,88-94).
Individual recombinant antigens could then be added, one by one, to
construct a series of highly sensitive (>95%) ELISA assays that also
could have acceptable specificity (>90%). At such a time, a two-tiered
testing procedure would make more sense. Furthermore, new genetic
markers for B. burgdorferi are being discovered and new PCR-like assays
will become easier to perform in the laboratory.

Additional progress, however, will be slow in Lyme diagnostics, until
we learn more regarding the biology of B. burgdorferi. In the course of
disease, long periods of remission are followed by acute symptoms that
may last for weeks or months. Therefore, basic research studies are
needed to evaluate the cyclical nature of the disease and the
idiosyncrasies of the organism, such as where it may reside in
extra-vascular spaces.

Science has progressed to the point where it effectively uses
techniques associated with molecular diagnostics and genetics, but some
of the traditional techniques may also be appropriate to study Lyme
disease. Tissue culture studies provide one level of understanding of
how the organism interacts with lymphocytes. The infection of research
animals, such as mice and dogs, using ticks with radio-labeled B.
burgdorferi, may provide information in a homeostatic environment,
where different types of cells and tissues can be studied. Progress for
better diagnosis and treatment, in this very complex disease, will come
through better knowledge of the spirochete B. burgdorferi.

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