Judy, do I now need to post all the citations about evasion of host
immune response by borrelia??  ;-D

They mutate, they coat themselves in host protein, they modulate the
immune system to make it more hospitable, and only 80 of the 300 or so
strains have their proteins included in the FDA approved WB kit.
Further, NSAID and antibiotic use is known to abrogate host immune
response, so you may never produce detectable antibodies to an active
infection.  Borrelia also shed their cell walls, further complicating
matters.

I could go on, and on and on...   <g>

I'm not saying this is what you have, I'm saying it's one of the
possibilities and you have a few of the highest risks in your hx.

Susan

I think that if the suspicion is high, you treat before you get the
results.  What's a little (actually, I'd go for 400mg per day for 4-6
weeks) doxy compared to a possible lifetime of mental and physical debility?

#41 is a non specific band, and it infuriates me that they chose it to
protect the vaccines they had coming out.  It's just flagellar protein,
that could mean gum disease (which should be treated too!).

When you order western blots, write on the slip "report ALL bands, not
just CDC specific ones."  Some of the bands they don't consider are
highly specific to B.b.

Here's a primer written by my friend, who was on the oversight committee
for the NIH studies, and had a letter published in the journal Science
refuting Steere's overdiagnosis publication:

Explanation of the Lyme Disease Western Blot
by
Carl Brenner
Inquiries about various issues relating to Western blot (WB) testing are
frequently posted to the Lyme disease discussion groups on the Internet.
Among the most commonly asked questions are:

    * What laboratory techniques are used to carry out the assay?
    * What exactly is being measured?
    * What is a "band"?
    * How are the results interpreted?
    * What are the CDC criteria for a "positive" test?

Although some of the medical jargon associated with immunology can be a
little overwhelming, the scientific principles behind these tests are
not difficult to grasp. The following article is offered as a primer in
the techniques and interpretation of Western blotting, and should help
most patients navigate their way through some of the medical and
scientific terminology associated with the assay.

First of all, it should be noted that the Western blot is usually
performed as a follow-up to an ELISA test, which is the most commonly
employed initial test for Lyme disease. "ELISA" is an acronym for
"enzyme-linked immunosorbent assay." There are ELISA tests and Western
blots for many infectious agents; for example, the usual testing regime
for HIV is also an initial ELISA followed by a confirmatory Western blot.

Both the ELISA and the Western blot are "indirect" tests -- that is,
they measure the immune system's response to an infectious agent rather
than looking for components of the agent itself.

In a Lyme disease ELISA,

   1. antigens (proteins that evoke an immune response in humans) from
Borrelia burgdorferi (Bb) are fixed to a solid-phase medium and
   2. incubated with diluted preparations of the patient's serum. If
antibodies to the organism are present in the patient's blood, they will
bind to the antigen.
   3. These bound antibodies can then be detected when a second
solution, which contains antibodies to human antibodies, is added to the
preparation. Linked to these second antibodies is an enzyme which
changes color when a certain chemical is added to the mix.

Although the methodology is somewhat complicated, the basic principle is
simple: the test looks for antibodies in the patient's serum that react
to the antigens present in Borrelia burgdorferi. If such antibodies
exist in the patient's blood, that is an indication that the patient has
been previously exposed to B. burgdorferi.

However, many different species of bacteria can share common proteins.
Most Lyme disease ELISA's use sonicated whole Borrelia burgdorferi --
that is, they take a bunch of B. burgdorferi cells and break them down
with high frequency sound waves, then use the resulting smear as the
antigen in the test. It is possible that a given patient's serum can
react with the B. burgdorferi preparation even if the patient hasn't
been exposed to Bb, perhaps because Bb shares proteins with another
infectious agent that the patient's immune system *has* encountered. For
example, some patients with periodontal disease, which is sometimes
associated with an oral spirochete, might test positive on a Lyme ELISA,
because their sera will react to components of Bb (like the flagellar
protein, which is shared by many spirochetes) even though they
themselves have never been infected with Bb. Therefore, some positive
Lyme disease ELISA results can be "false" positives.

To distinguish the false positives from the true positives, a more
specific laboratory technique, known as immunoblotting, is used.

    * The Western blot, which identifies specific antibody proteins, is
but one kind of immunoblot;
    * there is also a Northern blot, which separates and identifies RNA
fragments,
    * and a Southern blot, which does the same for DNA sequences.

In a Western blot, the testing laboratory looks for antibodies directed
against a wide range of Bb proteins.

   1. This is done by first disrupting Bb cells with an electrical
current and then "blotting" the separated proteins onto a paper or nylon
sheet. The current causes the proteins to separate according to their
particle weights, measured in kilodaltons (kDa).
   2. From here on, the procedure is similar to the ELISA -- the
various Bb antigens are exposed to the patient's serum, and reactivity
is measured the same way (by linking an enzyme to a second antibody that
reacts to the human antibodies).

If the patient has antibody to a specific Bb protein, a "band" will form
at a specific place on the immunoblot. For example, if a patient has
antibody directed against outer surface protein A (OspA) of Bb, there
will be a WB band at 31 kDa. By looking at the band pattern of patient's
WB results, the lab can determine if the patient's immune response is
specific for Bb.

Here's where all the problems come in. Until recently, there has never
been an agreed-upon standard for what constitutes a positive WB.
Different laboratories have used different antigen preparations (say,
different strains of Bb) to run the test and have also interpreted
results differently.

    * Some required a certain number of bands to constitute a positive
result,
    * others might require more or fewer.
    * Some felt that certain bands should be given more priority than
others.
    * In late 1994, the Centers for Disease Control and Prevention
(CDC) convened a meeting in Dearborn, Michigan [1] in an attempt to get
everybody on the same page, so that there would be some consistency from
lab to lab in the methodology and reporting of Western blot results.

Before we get to the recommendations that resulted from this meeting, we
need to understand one more facet of the human immune response. Many
patients have noticed that their Western blot report usually contains
two parts: IgM and IgG. These are immunoglobulins (antibody proteins)
produced by the immune system to fight infection.

   1. IgM is produced fairly early in the course of an infection, while
   2. IgG response comes later.

Some patients might already have an IgM response at the time of the EM
rash; IgG response, according to the traditional model, tends to start
several weeks after infection and peak months or even years later. In
some patients, the IgM response can remain elevated; in others it might
decline, regardless of whether or not treatment is successful.

Similarly, IgG response can remain strong or decline with time, again
regardless of treatment. Most WB results report separate IgM and IgG
band patterns and the criteria for a positive result are different for
the two immunoglobulins.

Finally, in setting up a nationwide standard for a positive WB, one
makes several assumptions --

   1. that all strains of Bb will provoke similar immune responses in
all patients,
   2. that all patients will mount a measurable immune response when
exposed to Bb, and
   3. that the IgG immune response will persist in an infected patient.

Unfortunately, none of these is always true. Therefore, a judicious
interpretation of Western blot results in a clinical setting should take
into account both

   1. the vagaries of the human immune response and
   2. the possibility that strain variations in Bb might produce
unusual banding patterns.

The CDC criteria for a positive WB are as follows:

    * For IgM, 2 of the following three bands: OspC (22-25), 39 and 41.
    * For IgG, 5 of the following ten bands: 18, OspC (22-25), 28, 30,
39, 41, 45, 58, 66 and 93.

How were these recommendations arrived at? The IgG criteria were taken
pretty much unchanged from a 1993 paper by Dressler, Whalen, Reinhardt
and Steere [2]. In this study, the authors performed immunoblots on
several dozen patients with well characterized Lyme disease and a strong
antibody response and looked at the resulting blot patterns. By doing
some fairly involved statistical analysis, they could determine which
bands showed up most often and which best distinguished LD patients from
control subjects who did not have LD. They found that by requiring 5 of
the 10 bands listed, they could make the results the most specific, in
their view, without sacrificing too much sensitivity. ("Sensitivity"
means the ability of the test to detect patients who have the disease,
"specificity" means the ability of the test to exclude those who don't.
Usually, an increase in one of these measures means a decrease in the
other.) The IgM criteria were determined in much the same fashion (by
different authors in different papers). Fewer bands are required here
because the immune response is less mature at this point. Several
studies have shown that

   1. the first band to show up on a Lyme disease patient's IgM blot is
usually the one at 41 kDa,
   2. followed by the OspC band and/or the one at 39.

The OspC and 39 kDa band are highly specific for Bb, while the 41 kDa
band isn't. That's why the 41 by itself isn't considered adequate.
Here's the rub, though: the CDC doesn't want the IgM criteria being used
for any patient that has been sick for more than about six weeks. The
thinking here is that by this time an IgG response should have kicked in
and the IgM criteria, because they require fewer bands, are not
appropriate for patients with later disease.

A number of criticisms have been offered of the CDC criteria since their
adoption in 1994.

   1. The first is centered on the CDC's failure to make any
qualitative distinction among the various bands that can show up on a
patient's Western blot.
      A number of Lyme disease researchers feel that different bands on
a WB have different relative importance -- that "all bands are not
created equal."
      For example,
          * many patients with Lyme disease will show reactive bands
at, say, 60 and/or 66 kDa. However, these correspond to common proteins
in many bacteria, not just Borrelia burgdorferi, and so are of limited
diagnostic usefulness, especially in the absence of other, more
species-specific bands. The band at 41 kDa corresponds to Bb's flagella
(the whip like organelles used for locomotion -- Bb has several) is one
of the earliest to show up on the Western blots of Lyme disease
patients. But for some reason it is also the most commonly appearing
band in control subjects. This may be due to the fact that many people
are exposed to spirochetes at some time in their lives and so their sera
might cross react with this protein.

          * On the other hand, certain other bands are considered
highly specific for Bb -- the aforementioned
                o 31 kDa band, for example, or
                o 34 (OspB) or
                o 39 or OspC (anywhere between 22 and 25).
            Also thought to be species-specific are
                o The 83 and
                o 94 kDa bands.
            Many Lyme disease scientists believe that any patient whose
IgG Western blot exhibits bands at, say, any three (or even two) of
these locations almost certainly has Lyme disease, regardless of whether
or not any other bands are present. They feel that these bands on a Lyme
Western blot are simply more meaningful than other, less specific ones
and that a rational interpretation of a WB result should take this into
account. Unfortunately, this does not often happen, and will happen even
less with the new CDC criteria.

   2. A second criticism of the CDC Western blot criteria is that they
fail to include the 31 and 34 kDa bands.
      This does indeed seem like an odd decision, since antibodies with
these molecular weights correspond to the OspA and OspB proteins of B.
burgdorferi, which are considered to be among the most species-specific
proteins of the organism.
      So why didn't Dressler et al. include them?
      Answer: These bands tend to appear late if at all in Lyme disease
patients, and did not show up with great frequency in the patients that
the Dressler et al. group studied (though they did show up sometimes).
As a result, they weren't deemed to have much diagnostic value and
didn't find their way onto the CDC hot list.
      However,
          * while the absence of either of these bands from a patient's
immunoblot result does not rule out Lyme disease,
          * their presence is hardly meaningless.
      Thus, many Lyme disease experts believe it is a serious mistake
to exclude these two antibody proteins from the list of significant
bands. The CDC's decision to do so seems particularly strange in light
of the fact that it is the OspA component of Bb that is being used as
the stimulating antigen in the ongoing experimental Lyme disease vaccine
trials. As one immunologist remarked shortly after the 1994 CDC
conference, "If OspA is so unimportant, then why the heck are we
vaccinating people with it?"

   3. Finally, it is important to keep in mind that no matter how
carefully the Western blot test is carried out and interpreted, its
usefulness, like that of all tests that measure B. burgdorferi
antibodies, is ultimately contingent on the reliability of the human
immune response as an indicator of exposure to B. burgdorferi. There are
several scenarios in which the lack of a detectable antibody response
may falsely suggest a lack of B. burgdorferi infection.
         1. First, it is well established that early subcurative
treatment of Lyme disease can abrogate the human immune response to B.
burgdorferi [3]. Although this is not thought to be a common phenomenon,
a recent comparative trial for the treatment of erythema migrans found
that a majority of patients who failed early treatment and suffered
clinical relapse were seronegative at the time of relapse [4]. Even
treatment for disseminated Lyme disease, in which the patient's IgG
immune response was previously well-established, can render a patient
seronegative after treatment despite post-treatment culture-positivity
for B. burgdorferi [5, 6].
         2. In addition, patients with Lyme disease may not test
positive for exposure to B. burgdorferi because their antibodies to the
organism are bound up in immune complexes [7]. Once steps are taken to
dissociate these immune complexes, free antibody can be detected;
however, this is not routinely done when performing serologic tests for
Lyme disease.
         3. Finally, an indeterminate number of patients with late Lyme
disease are simply seronegative for unknown reasons [8]. The actual
percentage of such cases as a proportion of all Lyme disease cases is
impossible to estimate, since most studies of late Lyme disease enroll
only seropositive patients, which tends to reinforce the circular and
erroneous notion that virtually all patients with late Lyme disease are
seropositive.
         4. It should also be noted that a positive Western blot is not
necessarily an indication of active Lyme disease. A patient's immune
response to B. burgdorferi can remain intact long after curative
treatment for a Lyme infection; therefore, the results of a Western blot
assay should always be interpreted in the context of the total clinical
picture.

Addendum by Joachim Gruber: Carl Brenner is one of 2 patients who sit on
the National Institute of Allergy and Infectious Diseases (NIAID)
Advisory Committee for Clinical Studies on Chronic Lyme (information
from Ramp S, The dirty truth behind Lyme disease research, Lyme Times
26,7, 1999).
REFERENCES
[1] Proceedings of the Second National Conference on Serologic Diagnosis
of Lyme Disease, October 27-29, 1994.
[2] Dressler F, Whalen JA, Reinhardt BN, Steere AC. Western blotting in
the serodiagnosis of Lyme disease. J Infect Dis 1993;167:392-400.
[3] Dattwyler RJ, Volkman DJ, Luft BJ et al. Seronegative Lyme disease:
dissociation of specific T- and B-lymphocyte responses to Borrelia
burgdorferi . N Engl J Med 1988;319:1441-6.
[4] Luft BJ, Dattwyler RJ, Johnson RC et al. Azithromycin compared with
amoxicillin in the treatment of erythema migrans. Ann Intern Med
1996;124:785-91.
[5] Häupl T, Hahn G, Rittig M, et al. Persistence of Borrelia
burgdorferi in ligamentous tissue from a patient with chronic Lyme
borreliosis. Arth Rheum 1993;36:1621-6.
[6] Preac-Mursic V, Marget W, Busch U, Pleterski Rigler D, Hagl S. Kill
kinetics of Borrelia burgdorferi and bacterial findings in relation to
the treatment of Lyme borreliosis. Infection 1996;24:9-18.
[7] Schutzer SE, Coyle PK, Belman AL, et al. Sequestration of antibody
to Borrelia burgdorferi in immune complexes in seronegative Lyme
disease. Lancet 1990;335:312-5.
[8] Liegner KB. Lyme disease and the clinical spectrum of antibiotic
responsive chronic meningoencephalomyelitides. (Abstract, 1996 LDF
Conference, Boston. MA)