Detection
Problems Resolved by Imaging with the Bradford Variable Projection
High Resolution Microscope
© 2004 Bradford Research Institute
Introduction
In the mid-14th century, Europe was
swept by a horrific catastrophe, known variously as the Bubonic Plague,
the Black Death or simply
the Pestilence. It is estimated to have killed in excess of 20 million
people, a third of the population of Europe at that time. It is believed
that half of the inhabitants of Paris died as a result of the plague.
Today we have learned to control the microorganism that caused the
great plagues of Europe and elsewhere but now we are beset by another "plague" that
is not as well known as that of 14th century Europe. The disease
was first recognized in the United States in the small New England
town of Lyme, Connecticut, and has since taken that name. Lyme disease
was first studied in 1975 by Dr. Allen Steere, following a mysterious
outbreak in that town of juvenile rheumatoid arthritis. The relationship
between rheumatoid arthritis and a disease of another name may not
at first be apparent but, as discussed more fully below, Lyme disease
has the ability to mimic many other diseases, making diagnosis extremely
difficult.
In 1982 the agent responsible for Lyme disease was discovered by Dr.
Willy Burgdorfer, isolating spirochetes belonging to the genus Borrelia
from the mid-guts of ticks infecting deer, other wild animals and dogs.
Spirochetes are spiral-shaped bacteria of very early origin in the
evolutionary scheme. The causative organism was named Borrelia burgdorferi
(Bb), after its discoverer. Since then, the number of reports of Lyme
disease have increased so dramatically that today, Lyme disease is
the most prevalent tick-borne illness in the United States.
The Centers for Disease Control (CDC) in Atlanta, Georgia, reports
that "there is considerable under-reporting" of Lyme disease,
maintaining that the actual infection rate may be 1.8 million, 10 times
higher than the 180,000 cases currently reported. Dan Kinderleher,
M.D., an expert on Lyme disease, stated that the number of cases may
be 100 times higher (18 million in the United States alone) than reported
by the CDC. It is estimated that Lyme disease may be a contributing
factor in more than 50% of chronically ill people.1
According to an informal study conducted by the American Lyme Disease
Alliance (ALDA), most patients diagnosed with Chronic Fatigue Syndrome
(CFS) are actually suffering from Lyme disease. In a study of 31 patients
diagnosed with CFS, 28 patients, or 90.3%, were found to be ill as
a result of Lyme disease.1
History of Lyme and Related Spirochetal Diseases
The discovery by Burgdorfer that Lyme
disease was caused by a spirochete placed it in a category of other
diseases known to be caused by spirochetes.
An example of such a disease is syphilis, the scourge of Europe for
hundreds of years. Arsenic and some of its compounds had been known
for quite some time as a highly successful and popular means of fatally
poisoning someone. Following the discovery of the Germ Theory of
Disease by Louis Pasteur (1822-1895), it was theorized that if arsenic
was toxic enough to kill, it may also be effective in killing the
organisms that cause disease. In the early 1900s, the German chemist-physician
Paul Ehrlich (1854-1915) developed a chemical treatment for syphilis.
By using a "shotgun" approach of trying hundreds of compounds
in an effort to find one that worked, Ehrlich discovered what became
known as Salvarsan or "606" after 606 compounds had been
tested. Salvarsan was an organic compound of arsenic and may be highly
toxic if not properly used. For his monumental discovery, Ehrlich
was awarded the Nobel Prize in 1908. Salvarsan may be considered
the first man-made antibiotic.2
Arsenic belongs to that column in the periodic table of chemical
elements known as the "Group V elements," also including
phosphorus, antimony and bismuth. See
Chart 1. (43KB .pdf)
Following the success of Salvarsan as a treatment for syphilis, other
compounds of antimony and bismuth were also prepared and tried against
spirochetes. Examples of these compounds include bismuth subcitrate,
bismuth subsalicylate (Pepto-Bismol), bismuth subgallate and many others.
An example of an antimony-containing antibiotic is Pentostam (an antimonial,
antimony sodium gluconate).3,4
A biological molecule known as ATP (adenosine triphosphate) supplies
energy to biological systems and does so through the high energy bonds
found in a chain of three terminal phosphate groups. One of the mechanisms
by which arsenic exerts its toxic effect is the substitution of phosphorus
by arsenic in ATP, since both arsenic and phosphorus lie in the same
column of the periodic table of chemical elements and have similar
chemistry. See Chart 2. (76KB .pdf)
When this substitution occurs, the molecule experiences immediate
hydrolysis, breaks down and is no longer functional as a source of
energy for the cell. Phosphorus, arsenic
and antimony are also found in this column of the periodic table (Group
V).5,6 (106KB .pdf)
What may be the first case of Lyme disease was noted about 1974 in
a 14-year old boy, taken to the hospital with extreme pains in the
muscles of his legs and unable to walk. This case, coupled with other
pertinent facts related to the boy and a highly classified US Government
laboratory conducting research on contagious animal diseases in this
same area is suggestive of a link between these two events. The Government
laboratory alluded to is found on Plum Island, just north of Long Island,
NY, and south of Lyme, Connecticut. Because of its secret nature, access
to the island was only by ferryboat and restricted to the Government
workers employed there. The 14-year old boy lived near the ferryboat
dock. Although not providing proof, these considerations are highly
indicative of a possible link between this research laboratory and
the subsequent outbreak in 1975 of an unknown disease involving juveniles
in Lyme, CT.29
History of Lyme Disease |
1900 |
Effective antisyphilitic, Salvarsan, (syphilis, a spirochete
disease) discovered by Paul Ehrlich, MD |
1908 |
Ehrlich awarded Nobel Prize for the arsenic-containing compound
to treat syphilis |
1952
to date |
Highly classified US Government animal disease research laboratory,
Plum Island, in close proximity to Lyme, CT |
1974 |
First Lyme symptoms, 14-year old boy, Lyme, CT |
1975 |
Lyme disease first recognized by Allen Steere, MD, in Lyme, CT |
1982 |
The causative Lyme spirochete was discovered by Dr. Willy Burgdorfer |
1983 |
Borrelia burgdorferi was named after Dr. Willy Burgdorfer |
2003 |
The Bradford Research Institute's High Resolution Microscope
imaging of Lyme spirochete and cyst forms |
2004 |
The Bradford Research Institute (BRI) developed BismacineTM ,
an
injectable form of bismuth, shown effective against the spirochete
and cyst forms |
|
© 2004 BRI |
Etiology and Difficulty of Treatment
The first step in being able to treat any disease is to learn the cause
(etiology) of that disease. Once the cause of Lyme disease was known,
it would seem that a treatment modality would soon follow and the
problem would be solved. Unfortunately, as history has shown, this
was not to be the case. As more was learned about the causative agent,
namely, the spirochete Borrelia burgdorferi, it became obvious that
this organism was unlike any that had been previously studied. It
is one of the largest of spirochetes (0.25 x 25 m). Spirochetes in
general are difficult to treat for several reasons; they have the
ability to burrow into or between cells and hide, gaining protection
from the immune system. Both Bb and Treponema pallidum, the causative
agent for syphilis, have highly unusual outer membranes and the molecular
architecture of these membranes is responsible for their ability
to cause persistent infection.
Bb also has a three-layer cell wall, helping to determine the spiral
shape of the spirochete. This distinctive cell wall resembles those
of Gram-negative bacteria, although Bb does not stain Gram-negative
but is stained by silver stains (containing silver nitrate). This characteristic
may be related to the purported treatment of Lyme disease by colloidal
silver.
Another unusual structural feature is a single flagella, attached to
each end of the spirochete, running the length of the organism and
surrounded by it. This feature is significant in relation to immune
protection since most bacterial flagella are highly antigenic. Still
another difference in Bb structural architecture is a clear gel-like
coating surrounding the bacteria, giving it protection from the immune
system.28 See Photo 1. Borrelia
burgdorferi Spirochete (17KB .pdf © BRI 2004)
The DNA of Bb is arranged in a different
manner than in other bacteria, lying along the inside of the inner
membrane, resembling a net just
under the skin. The bacteria spelicates specific genes, inserts them
into its own cell wall and then pinches off that part of the cell membrane,
releasing it into the surrounding medium. This fragment of the spirochete
membrane with incorporated DNA is known as a "bleb." It
is not understood why this strange event occurs or what advantage it
gives the organism but some studies suggest that the function of blebs
is to bind IgM antibodies, thereby protecting the organism from the
immune system. See Photo
9, (152KB .pdf) courtesy of Microbiol. & Immunol.
26 (3) (1982).
The spirochete is typically observed in three different forms utilizing
the Bradford Variable Projection Microscope (BVPM).
Bradford Microscopy of a normal spiral form of spirochete, length of
approximately 25 m with evenly spaced blebs along its membrane. (62
KB .pdf, Darkfield-Phase, 10,000X, ©BRI 2004)
Bradford Microscopy of the elongated
bleb form described above, by doubling back on itself, forms a circle
of blebs. See Photo 3.
(89KB .pdf, Darkfield-phase, 10,000X, ©BRI
2004)
Bradford Microscopy of the elongated
form doubled back on itself, forming close-packed multiple clusters
of figure 8s (convolutions),
typically observed inside a B-cell, but may been seen isolated. (28KB
.pdf, Phase, 18,000X, © BRI 2004)
Bradford Microscopy of a cyst form
developed inside a B-cell, without the clustered spiral form of the
spirochete. (89KB .pdf, Phase-Darkfield, 10,000X, © BRI
2004) With clustered spiral
form of spirochete
(67KB .pdf, Phase-Darkfield,10,000X, © BRI
2004)
Bradford Microscopy
of a cyst form inside a basophil. (74KB .pdf, Darkfield-Phase,
12,000X, © BRI
2004)
Bradford Microscopy
of a cyst form inside an eosinophil. (70KB .pdf, Darkfield-Phase,
10,000X, © BRI
2004)
Scanning
electron microscopy of blebs on spirochete membrane. (61KB .pdf,
Scanning Electron; Microscopy)
The cell division time of Bb is very long compared to other bacteria.
A typical cell wall reproduction time for Streptococcus or Staphylococcus
is less than 20 minutes, while the total reproduction time of Bb is
from 12-24 hours. Most antibiotics inhibit the formation of cell
walls and are effective only when the bacteria are dividing with the
formation of new cell wall. With the slow replication time of Bb, an
antibiotic would have to be present 24 hours a day for one year and
six months to be present during the cell wall reproduction period.
There are basically two mechanisms by which Bb can survive within the
host and remain for long periods of time, unknown by the victim. Because
of these processes, a person infected by Bb can remain unsymptomatic
for long periods of time and then suddenly, without warning, begin
to experience symptoms once again. One of these mechanisms involves
the invasion of tissues by the spirochete. The tip of the organism
has the ability to bind to cells, spin and twirl until it stimulates
the cells own enzymes to digest a part of the membrane, finally allowing
entry. Once inside, the spirochete results in either the death of the
cell or takes up residency within. It may lie dormant for years, protected
from both the immune system and the action of antibiotics.
Experiments have shown that if a culture of Bb is placed under conditions
of nutrient deprivation or starvation, it senses that it cannot survive
in a metabolically active state and generates what are known as "cysts" or
small sacs attached to the organism by slender threads. Cysts contain
immature spirochetes in a metabolically inactive form. Eventually they
break off from the parent body and either remain lodged in tissues
or enter the blood where they are sensed as foreign antigens by eosinophils
(a type of WBC) and phagocytized. Eosinophils release granules of positively
charged basic protein, attaching to the normally negative surface of
cells. They attempt to destroy the invading foreign bodies (cysts)
but have little success. See
Photo 9. (152KB .pdf, Scanning electron microscopy
of the spirochete cyst form)
When a spirochete attacks a B-cell, it attaches the tip to the surface,
spins and twirls until it enters, then multiplies inside until the
B-cell bursts. Some of them become coated with fragments of B-cell
membrane and escape detection by the immune system by masquerading
as a B-cell. Most of the antigenic proteins in Bb (that in other bacteria
mark the microorganism for destruction by the immune system) are found
on the inside of the inner membrane where they cannot contact those
WBC that detect invaders.
Experiments have shown that Bb can rather quickly change surface antigens
so that antibodies made against one strain are effective in killing
that strain but a second strain having different surface antigens will
take up residence in a different tissue where it escapes detection
and survives. For these reasons and others it becomes apparent that
this particular spirochete has evolved guises and biological techniques
to guarantee its survival and thwart any attempts to circumvent it.7
Distinguishing Characteristics
of Borrelia burgdorferi |
Internal Flagella |
Cyst Formation |
Glycoprotein Coat |
Destruction of B-Cells |
DNA Net Arrangement |
Camouflage as B-Cells |
Bleb Formation |
Internal Antigenic Proteins |
Prolonged Replication Time |
Surface Antigen Transformation |
Cellular Invasion Ability |
Spiral Shape |
|
© 2004 BRI |
Life Cycle of Borrelia burgdorferi and Related Tick
The life cycle of Bb is related to the life cycle of the associated
tick (usually Ixodes scapularis). The tick has four stages in its
two-year life cycle: egg, larva, nymph and adult. The tick usually
acquires the spirochete during its larval stage, when it feeds on
small animals such as rodents or birds. The tick then becomes the
host for the spirochete. The bacteria resides in the digestive tract
of the host for its next nymph and adult stages during which it is
passed on to other animals and/or humans. It has been learned that
Bb may also be carried and transmitted by fleas, mosquitoes and mites.(See
illustration, Life Cycle of a Lyme Disease
Tick. 48KB .pdf)
Signs and Symptoms of Infection
The first recognizable symptom following a tick bite is the development
of a rash at the site within 7 to 10 days. The rash expands with
an area of central clearing. Other symptoms may include low-grade
fever and/or headache. The rash and early symptoms clear within 3
to 4 weeks. Multiple secondary rashes may occur following this time
period. Bouts of arthritis, usually involving large joints, especially
the knee, are very common. Arthritis attacks usually resolve within
3 to 4 years with or without treatment.
Early neurological complications include Bell's palsy, meningitis
and encephalitis. Sub-acute symptoms may include cognitive deficits,
mood and sleep disturbances, persisting for more than 10 years. One
of the most common symptoms is intense fatigue. Additional symptoms
may include memory loss, poor coordination, slurred speech, poor concentration,
unusual depression, burning, stabbing pain, tremors, anxiety, swollen
glands and tinnitus.
Some Lyme Disease Signs and Symptoms
Intense Fatigue
Memory Loss
Burning/Stabbing Pain
Tremors
Joint Pain/Swelling/Stiffness
Shortness of Breath
Poor Coordination
Anxiety
Slurred Speech
Swollen Glands
Chills and/or Fever
Nausea/Vomiting
Rash
Muscle Cramps
Sudden Mood Swings
Headaches/Migraines
Poor Concentration
Light Sensitivity
Unusual Depression
Tinnitus
© 2004 BRI
Other conditions most commonly seen with Lyme disease include Alzheimer's
disease, amyotrophic lateral sclerosis (ALS), chronic fatigue syndrome
(CFS), fibromyalgia, irritable bowel syndrome, lupus, rheumatoid arthritis,
scleroderma, multiple sclerosis (MS), Parkinson's disease and
various autoimmune disorders.8
Most Common Diseases Associated with Lyme
Alzheimer's Disease
Polymyalgia rheumatica
ALS
Reflex sympathetic dystrophy
Bell's Palsy
Rheumatoid Arthritis
Chronic Fatigue Syndrome (CFS)
Scleroderma
Fibromyalgia
Syphilis
Irritable Bowel Syndrom
Multiple Sclerosis
Lupus
Parkinson's Disease
Depression
Autoimmune Disorders
Middle Ear Pressure
Tinnitus
Vertigo
Rheumatoid Arthritis
© 2004 BRI
Ticks may carry more than one infectious organism and, for this reason,
a person infected by Bb may also be infected by other microorganisms,
leading to symptoms of those diseases as well. Examples of organisms
commonly occurring with Bb include Babesia microti,25 Ehrlichia chafeensis,26
E. equi, Mycoplasma pneumoniae, Chlamydia pneumoniae, Bartonella henselae
and Rickettsia rickettsiae. The presence of multiple symptoms of several
different diseases makes diagnosis and treatment of Lyme disease much
more difficult.8
Therapy for Lyme Disease
Antibiotics
When conditions become adverse for its survival, Bb produces cysts
containing the DNA defining the organism intended for future generations
but surviving in a metabolically inactive state. In this state there
is no cell wall generation and no way an antibiotic can damage the
organism.9
It has been found that tetracycline can inhibit cyst formation and
damage the envelope of cysts. It is also believed that bismuth compounds
can enter cysts through the cyst wall. In addition, the prolonged replication
rate, mentioned previously, protects the organism from cell wall damage
by most antibiotics.10
Antibiotics Commonly Used in Lyme Treatment
Tetracycline
Salvarsan
Amoxacillin
Cefuroxime
Doxycycline
Clarithromycin
Flagyl
Metronidazole
Cefotaxime
Ceftriaxone
Azithromycin
Penicillin
Imipenem
Benzathine Penicillin
Cefdinir
Rocephin
Tinidazole
Trimethoprim
Cipro(floxacin)
© 2004 BRI
Oral Salt Therapy
Certain white blood cells (WBC) display several distinct mechanisms
that may be employed for the purpose of killing invading microorganisms.
One of these deserves particular attention in relation to killing
the causative agent of Lyme disease, namely, the spirochete Borrelia
burgdorferi.
Neutrophils (a class of WBC) contain two essentially different types
of storage granules, peroxidase-positive granules and peroxidase-negative
granules. Peroxidase-positive granules contain myeloperoxidase, an
enzyme that uses hypochlorous acid (HOCl) in conjunction with hydrogen
peroxide, providing a source of nascent (atomic) oxygen for the purpose
of killing invading microorganisms.11
Peroxidase-negative granules contain a family of large polypeptides
(11 to 19 kDa) (Dalton, the unit of molecular weight) known as the
cathelicidins or, in humans, hCAP-18. A segment of this larger or precursor
protein (also known as a Bacteriacidal Permeability-Increasing (BPI)
protein) is proteolytically removed by the enzyme elastase found in
peroxidase-positive granules. The better-known substrate of elastase
is the elastic protein elastin, found in skin and other tissues requiring
elasticity. By incorporating elastase inhibitors into skin creams,
attempts are made to inhibit the activity of this enzyme, thereby decreasing
the ageing of skin. In Lyme therapy there is an advantage (described
below) to increasing the activity of this enzyme, thereby stimulating
the natural antimicrobial system. These short peptides, ranging from
12 to 100 amino acids, have the ability to assemble into larger units
that form pores in the membrane surrounding microorganisms, thereby
increasing the permeability of those membranes. In humans, one of these
antimicrobial peptides has been dubbed LL-37.11 See
photo of a neutrophil granule precursor antimicrobial protein and
peptide, 31KB .pdf, courtesy
of Blood 96 (8)
2000.
Both of these proteins, the cathelicidin and elastase, meet in the
phagocytic vacuole, the cytoplasmic chamber in which resides the phagocytized
microorganism. Within this chamber, elastase removes a short peptide
capable of forming a molecular pore in the surface membrane of the
microorganism. The pore formed from a group of the cathelicidins allows
the efflux of potassium ions from the organism, resulting in swelling
and eventual lysis.12
Research has shown that, of all the proteins in neutrophil granules,
the only protein capable of releasing the cathelicidin active peptide
is elastase.13 It has been demonstrated that the activity of elastase
is enhanced by an increased salt concentration.14 Through oral salt
(12 g per day, see Chart 12), combined with large doses of vitamin
C, the indirect killing ability of elastase is dramatically increased.15
Increasing the sodium concentration surrounding the spirochete may
also facilitate cell killing by allowing sodium ions to enter the spirochete
through the pore created by the antimicrobial peptide. An increased
intracellular sodium concentration, combined with a decreased potassium
concentration, leads to spirochete death. The exact mechanism by which
the human cathelicidin LL-37 kills Bb is unknown. See Chart 8, Oral
Salt Therapy for Lyme Disease. (58KB .pdf)
Colloidal Silver
It is believed that colloidal silver consisting of small clusters of
silver atoms in the elemental form may be effective in eradicating
Bb. If true, this action may be explained by the known ability of
the Bb spirochete to bind silver, resulting in a brown/black stain.16
Bee Venom
Bee venom is a mixture of enzymes that digest most if not all of the
various kinds of biological material. Of particular value in the
treatment of Lyme are the proteolytic enzymes, those that digest
protein. It is believed that the proteolytic enzymes in bee venom
are capable of digesting the protein coating or shell of Bb cysts.17
Bee venom also contains a number of potent peptides, responsible for
having a strong inhibitory effect on Bb. When the spirochete is inhibited
it does not multiply and is vulnerable to the host's own immune
system and other medications.
Herbal Therapy
Cat's Claw
An herbal commonly used in the treatment of Lyme disease is Cat's
Claw (Uncaria tomentosa), native to Peru and used for centuries by
that South American culture. Traditional Cat's Claw contains
chemical antagonists to the immune system known as tetracyclic oxindole
alkaloids (TOASs). Some Cat's Claw products and preparations
contain only the pentacyclic oxindole alkaloids (POAs. superior) for
stimulating the immune system. Some are standardized for the POAs they
contain.18
The results of research on Cat's Claw products containing POAs
(Samento, commercially-available), demonstrate powerful immune system
modulating and stimulating properties, along with pronounced anti-inflammatory,
antioxidant, and anti-infectious effects. Cat's Claw also contains
quinovic acid glycosides—compounds with strong natural antibiotic
properties.1
Artemesia
A second herb that has been used in Lyme therapy is Artemesia annua,
shown effective against Babesia, one of the more common infections
accompanying Bb in Lyme patients.19
Bradford Research Institute/Ingles Hospital Therapy
The Bradford Research Institute (BRI)/Ingles protocols includes two
antibiotics, Ciprofloxacin and Doxycycline. Also included are one
or both of two new bismuth-containing compounds developed by BRI,
injectible Bismacine-C and Bismacine-N. These new therapeutic agents
are currently being evaluated with Lyme patients in the BRI/Ingles
Hospital, Tijuana, B.C., Mexico.
Oral supplementation for pain includes the following regimen:
4-5 g buffered Vitamin C, 2x/day
5 Inflazyme Forte™ (4000 IU Pancreatin) tablets 2x/day
3 Oxy-5000 Forte™ 2x/day
50 mg Magnesium Aspartate tablet one 2x/day
Basic Elemental Minerals™ 2x/day
Summary of Lyme Disease Therapy
Antibiotics: Includes Tetracycline,
Amoxacillin, Cipro, Penicillin and Doxycycline
Oral Salt Therapy: The
enzyme elastase, found in neutrophils, is stimulated by high salt
concentrations
to remove a polypeptide LL-37
from the
precursor protein CAP-18. A group of the polypeptides assemble into
a pore and becomes imbedded into the outer membrane of the infectious
microorganism, allowing potassium and other ions to escape, thereby
killing the organism.
Coloidal Silver: Small clusters
of elemental silver atoms that bind to the spirochete, phagocytized
by PMNs.
Bee Venom: Contains proteolytic
enzymes that digest the outer coating of cysts. Also contains polypeptides
that inhibit spirochete growth
and reproduction.
Herbal Therapy: Includes fractionated
Cat's Claw, an immune system stimulant and Artemesia, effective in
combattin Babesia, a disease
associated with Lyme.
Dioxychlor, SulfoximeTM, Bismacine-CTM: Bradford
Research Institute/Ingles Hospital Therapy
© 2004 BRI
Bradford High Resolution Microscopy
As of this writing, the Bradford Research Institute/Ingles Hospital
has 100% confirmation between Lyme morphology obtained utilizing
the Bradford High Resolution Microscope and the Bowen fluorescent
antibody test, 36 patients with positive correlation and 3 controls
with negative correlation.
Rheumatoid Arthritis
In Lyme patients at the Bradford Research
Institute/Ingles Hospital, it has been observed that patients with
rheumatoid arthritis have
dramatically improved with the Bismacine treatment along with a broad-based
antimicrobial treatment (Sulfoxime™, Dioxychlor®).
In an integrative medical center, a specific treatment protocol is
tailored to the individual needs, based on the concurrent assessment
and diagnosis of functional pathology, organic pathology and the contributory
risk factors of stress and toxicities. Based on the above assessments,
an integrated treatment protocol is developed to meet the specific
patient needs.
Detection of Infection
Fluorescent
Antibody Test
In this test, antibodies to selected antigens on the Lyme causative
organism (Bb) are chemically (covalently) attached to a chromophore,
an organic chemical that fluoresces when irradiated by ultraviolet
light. When the test is made, blood from the patient is mixed with
the fluorescent antibody preparation. If Lyme antigens are present
(during infection with Bb), the antibodies complex with and bind to
the antigens present in the blood. Under ultraviolet irradiation, these
molecules fluoresce, revealing the presence of Lyme antigen. This test
is superior to other tests for the presence of Lyme disease and is
more reliable for indicating an infection with Bb.20
PCR (Polymerase Chain Reaction) Test
The PCR test is very sensitive in revealing the presence of a minute
amount of DNA, in this case the genetic material of Bb, indicating
an infection. The test involves the amplification or multiplication
of small amounts of DNA by supplying all of the requirements for
the replication of DNA. The units of DNA (nucleotides) are provided,
the enzyme for performing the replication is provided as well as
any other required substances. If DNA from Bb is present, it will
be amplified so that it may be detected by conventional means. Known
nucleotide sequences of Bb are compared to those revealed by the
sample. If similar, a positive identification of Bb is made.21
ELISA (Enzyme-Linked Immuno-Sorbant Assay)
The mechanism of the ELISA test is in some ways similar to the fluorescent
antibody test described above. In the ELISA test, the antibody made
by the infected person against Bb antigens as a result of infection
is covalently bound to (labeled with) an enzyme, typically horseradish
peroxidase. The commercially prepared and purified antigen (from
Bb) as a solution is allowed to bind to the surface of small wells
formed in a plate of polystyrene (commercially available). The wells
are then contacted over a specified time and at a specified temperature
with the enzyme-labeled antibodies (made by the patient against Bb
antigens during the course of the infection and present in serum).
The plate is then gently washed to remove any unbound proteins. The
substrate for the enzyme is provided and the plate is allowed to
develop for a specified time and at a specified temperature. If antibodies
against Bb antigens are present, the covalently attached enzyme will
act on the substrate provided and result in a color change. Following
incubation, the color changes are read and indicate the presence
of antibody, and therefore Bb antigen. This is a poor assay with
marginal sensitivity for Lyme. 22
Western Blot Test
The Western Blot test involves two electrophoretic steps in two different
directions, followed by the application of antibody for specificity.
In a typical procedure, serum from a Lyme patient is used as the
sample protein solution in slab-gel electrophoresis on polyacrylamide
gel (PAGE). The gel slabs containing the separated proteins are then
employed in transverse electrophoresis using thick, carbon block
(graphite) or platinum foil electrodes. Sandwiched between the gel
slab and the electrodes are several layers of blotting paper soaked
in electrically-conducting buffer (salts). The proteins are absorbed
(blotted) onto a membrane of nitrocellulose for subsequent binding
by commercially-available Bb antibody. Following the binding of specific
antibody, the nitrocellulose paper is gently washed free of extraneous
protein, retaining only the insoluble antigen-antibody complexes.
These complexes are then stained by any of a variety of protein dyes
and dried. The appearance of protein bands indicates the presence
of Bb antigens in the serum. The Western Blot test has a high percentage
of false negatives and should not be used to assess Lyme disease.23
The CDC Guidelines state that the ELISA test and the Western Blot test
are plagued with false negatives and are not to be used to exclude
diagnosis of Lyme disease.27
Detection of Borrelia burgdorferi (Bb)
Fluorescent Antibody Test: Antibodies to Bb are covalently coupled
to a fluorescent organic chemical and added to the patient's
blood on a microscope slide. Antibodies bind to antigens found on Bb
(spirochete or cyst form) and fluoresce under ultraviolet light, revealing
the presence of Bb. Most accurate.
PCR: DNA from Bb is allowed to replicate, thereby increasing the amount
present to enable a sequence determination to be made. The sequences
are compared to the known sequences of Bb. Test unreliable.
ELISAL: Antibodies to Bb are covalently coupled to a specific enzyme
and allowed to bind to Borrelia antigens in the presence of the enzyme
substrate. Enzyme activity results in a color change, revealing the
presence of Bb antigens.
Western Blot: The slab-gel electrophoresis of Lyme patient serum separates
Bb protein antigens. A second transverse electrophoresis carries the
antigens into a nitrocellulose membrane where they are revealed by
the application of specific antibody and staining. Gross false-negatives.
Bradford High Resolution Microscopy: Both
the cyst and spirochete forms in the three different morphologies are
easily identified with resolutions
less than 0.1 micron with concurrent magnification of 10,000x utilizing
dark-field and phase contrast modes.
Comparison of Tests: High
Resolution Microscopy is the most reliable test. PCR, Western Blot
and ELISA are the least reliable with up to
80% false-negatives (CDC Guidelines).
© 2004 BRI
Comparison of Detection Methods
The Centers for Disease Control (CDC)
in Atlanta, Georgia, has issued guidelines for Lyme patients, advising
them of a recommended protocol
in attempting to establish whether Lyme disease is present or not.
Doctors have been instructed by these guidelines to obtain an ELISA
test first, which, under the best circumstances, identifies only
40-50% of those who actually have Lyme disease. An ELISA should NOT
be used as a screening test due to the unreliable results. The guidelines
then state that, if the ELISA test is positive, doctors are to perform
the Western Blot test. This procedure allows many cases of Lyme disease
to be missed, therefore patients are not being identified or properly
treated. The CDC guidelines also state which specific bands on a
nitrocellulose strip are to be used in considering a test positive.
When the list of bands was developed, certain bands specific for
Lyme disease were not included. When these bands are positive, they
confirm exposure to the causative organism, but it is mistakenly
reported to the doctor and patient as a "negative test." Many
borderline tests are reported to patients as being negative and many
positive tests are reported to be "false-positive" because
doctors are not familiar with reading test results, nor with the
multiple symptoms that can occur in a person with Lyme disease.24,27
Charts 11, 12 and 12-A are typical Integrative Treatment Protocols
for Lyme patients.
Bradford Research Institute/Ingles Hospital Protocol for Lyme Disease
Chart 11: Daily Intravenous Infusions
Infusion I (Nutrient, Antioxidant,
Antimicrobial, 3-hour drip)
The following in 250 cc Saline:
10 cc DMSO
25 g Vitamin C
10 cc NAC
10 cc Taurine Plus™
5 cc Biorizin™
2.5 cc Multivitamin Combination
1 g EDTA
Infusion II in 100 cc saline, 1x/day,
10 cc Dioxychlor, over 30 minutes
Infusion III 2 cc each Bismacine-C™, Bismacine-N™, 2x/day,
1 hour
Infusion IV Sulfoxime™ (Antimicrobial) 1-3x/week, 200 cc, 20
min. drip
Infusion V Vitamin C 75 g in 250 cc saline, 2-hour drip, 1x/day
© 2004 BRI
Chart 12: Daily Oral Program
I. Vitamin C, 5 g 2x/day (controlled
release)
II. Inflazyme™, 5 tablets,
3x/day, 30 minutes before meals
III. Oxy-5000™, 3 tablets,
3x/day
IV. Magnesium Aspartate, 50 mg, 2x/day
V. Calcium (Osteo Synergy™),
50 mg, 2x/day
VI. Potassium, 50 mg, 2x/day
VII. Glutathione, 20 mg, 2-3x/day
VIII. Beta Carotene, 25,000 IU, 2x/day;
Vitamin E, 400 IU, 3x/day; Vitamin B12 Compl., Caps., 1-3x/day; Selenium,
200 mcg, 1-3x/day; Trace
Minerals, 1-3x/day
IX. Bowel Protocol (Overview)
1. Ultra-Micro-Plex™, 4 tbs in 4 oz. water + pinch of salt, 3x/day
2. Rectal Implant, 1 tsp Ultra-Micro-Plex in 4 oz. salt water, 1-2
x/day morning and night
3. Coffee Enemas, 1x in morning
4. Chamomile Enemas, 1x in afternoon
5. HCI Protocol as Required
© 2004 BRI
Chart 12A
X. Liver Protector, Hepatrope, 1-3x/day
XI. Glandular Support, Thymus 1-3x/day, Adrenal 1-3x/day, Thyroid,1-3x/day
XII. Homeopathic Remedies as Indicated
XIII. NeuroRecovery™, caps., 1-3 x/day
XIV. Oral Salt Treatment (Optional) 12 grams of Salt, spaced throughout
the day
XV. Artemesia
XVI. Samento (Cat's Claw)
XVII. Homeopathic Kidney Drainage
XVIII. Homeopathic Lymphatic Drainage
XIX. Colloidal Silver
© 2004 BRI
The Bowen Research & Training Institute, Palm Harbor, Florida,
is FDA-licensed to perform tests in which spirochetes in various forms
can be detected and photographed from tissue and blood samples. They
are also able to identify several strains of Babesia25 and Ehrlichiosis.26
This laboratory uses the fluorescent specific antibody test for detecting
Bb.20
Charts C, D, E and F summarize the basic concepts that have been presented,
including the best methods for the diagnosis and detection of Lyme
disease.
Chart C: Long-Standing Controversy Surrounds Lyme Disease
· There is no approved test
for Lyme disease, specifically ELISA, Western Blot and PCR.
· Fate of spirochetes after entering the human system is not totally
known with certainty.
· May enter B-cells, other WBC or a variety of tissues and organs.
· Professionals admit they do not know where the spirochete goes, where
it hides or how it may be detected.
·
NOTE: The Bradford High Resolution Microscope blood imaging has revealed
the location of the spirochete and cyst form in the B-cells, eosinophils
and basophils.
· Clinical evidence has revealed the associated Lyme spirochete with
elevated PSA, rheumatoid arthritis, CFS, MS, fibromyalgia and diabetes.
© 2004 BRI
Chart D: Difficulties in Diagnosis and Detection
· The Centers for Disease Control
(CDC) indicates that the number of Lyme cases may be in excess of 1.8
million.
· Other experts indicate that the figure may be in excess of 18 million
in the US alone.
· Lyme disease is difficult to diagnose because it mimics many other
diseases and symptoms.
· Poor detection, up to 80% false negatives using:
· ELISA (Immunological, patient's antibodies, color indicator)
· Western Blot (Electrophoresis of patient's antigens, application
of specific antibody)
· PCR (Amplification of spirochete DNA, sequence comparison)
· As a result, most Lyme patients today go untreated.
© 2004 BRI
Chart E: Lyme Disease Out of Control
· An estimated 18 million cases
in the US alone, many more worldwide.
· With poor diagnosis and treatment, there is little hope for a successful
resolution.
· Available treatment modalities are only partially successful; the causative
organism is masked in lymphocytes, eosinophils, basophils and tissues.
· One year ago, in the Bradford Research Institute/Ingles Hospital, Tijuana,
Mexico, 1 in 20 patients had Lyme disease. Today, the incidence is
in excess of 14 in 20.
Chart F: Solution to Detection Problem
· The Bradford High Resolution
Blood Morphology imaging of both Lyme spirochete and cyst forms have
proven to be highly accurate.
· The various cyst forms are found in B-cells, eosinophils, basophils,
with and without the spirochete.
· The detection of Lyme disease by the Bradford High Resolution Microscope
is highly correlated with the Fluorescent Antibody Test (FDA-licensed
Bowen Laboratories, Florida)
Discussion
The causative organism of Lyme disease
has developed the ability to not only disguise itself but to circumvent
the immune system in many
ways unparalleled by any other bacteria. Lyme disease has a reputation
of being extremely difficult to detect and diagnose with certainty,
leading many to believe they do not have the disease when, in fact,
they do. The Bradford Research Institute has made significant progress
in both the detection and treatment of Lyme disease, however, there
is at the present time no cure or "magic bullet" for
Lyme disease, implying that much additional research is greatly needed
to suppress this new alarming bacterial outbreak. The Lyme epidemic
has presented us with both a challenge as well as an opportunity
to resolve not only Lyme, but a number of associated immunological
and infectious conditions, stresses and toxicities.
Note: The Bradford Research Institute is seeking additional researchers
to participate in The International Metabolic Research and Development
Project (FDA-registered 1979), utilizing the Bradford Variable Projection
Microscope. The Bradford Variable Projection Microscope blood imaging
for functional assessments looks at 72 blood morphologies correlating
with 111 risk factors. To qualify, the researcher must have a "scope
of practice to diagnose and treat" in order to comply with the
established criteria. For more information we may be contacted at:
Phone: (619) 429–8200, (800) 227–4473. Fax: (619) 429–8004,
email: drbradford@americanbiologics.com. Websites: www.bradfordresearchinst.org and www.americanbiologics.com.
The authors wish to acknowledge Ann Marie Dixon, MBA, ND, Prof. of
Medicine, Capital University of Integrative Medicine, Washington, DC,
whose invaluable contribution to this manuscript is greatly appreciated.
©
2004 Bradford Research Institute. May be reproduced with written permission
and credit.
Correspondence:
Robert W. Bradford
Bradford Research Institute
1180 Walnut Avenue
Chula Vista, California 91911 USA
(619) 429–8200
drbradford@americanbiologics.com
http://www.americanbiologics.com
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