Lyme test book excerpt Part 3:
Just as some Tick-Borne-Relapsing-Fever patients have reported bull’s-eye rashes, some Lyme patients have reported relapsing febrile states similar to Tick-Borne-Relapsing-Fever patients.
All Borrelia bacteria evolved from the same ancient ancestors. That means that all of the Borrelia species are linked by genetics. Ignoring those similarities has led to many unnecessary squabbles by clinicians over what defines “Lyme disease” as a separate disease, and how scientific semantics keeps us away from having discussions about Tick-Borne-Relapsing -Fever Borrelia species.
When talking about Lyme disease testing, why is it important to classify Lyme disease as part of a larger group of similar Borrelia pathogens?
The answer: All Borrelia infections are bad, so why not have an inclusive test that that includes all species of pathogenic Borrelia. I once commissioned Gene-Link to make an all inclusive PCR test for all Lyme species and pathogenic Relapsing Fever species. The cost would have been about $12,000
Any test that is specific for detecting B. burgdorferi, is mostly useless for new variations of Borrelia that cause Lyme-Like disease such as what is being seen in Missouri. Or the newly emerging species of Lyme disease like B.kurtenbachia or B. carolinensis and 12 others and more to come.
In Missouri most “Lyme-like-Disease” is caused by Borrelia lonestarri. The current Lyme tests are not as sensitive to B. lonestarri, but the symptoms and consequences are identical. While health departments and clinicians argue over names and definitions, patients are being misdiagnosed and ignored and worst of all told they don’t have true Lyme disease and are sent home with nothing but their symptoms.
Categories of Lyme Tests
There are five main methods of testing for Lyme disease: *
1. Antibody Tests: (The Elisa and Western Blot serology tests are most commonly used.)
2. PCR Tests: Bacterial DNA detection by polymerase-chain-reaction test (PCR)
3. Live culture of the bacteria from the body
4. Antigen Detection Tests: Finding particles and proteins of the bacteria in blood, CSF, urine, or tissue samples.
5. Direct observation by microscope: This can employ the use of biopsy and stain, or
centrifuged blood, spinal fluid, and urine.
*Other indirect tests such as T-cell activation test, Immune Fluorescence Tests, the Lyme Urine Antigen test, and the Immune Sera Culture Assay will not be explained in this booklet.
ANTIBODY TESTS
Antibody tests are by far the most commonly used tests for Lyme disease, and unfortunately one of the most misunderstood tests by physicians. These tests are indirect measurements of your body’s response to the bacterial pathogen.
Antibody serology tests are most often employed not for their accuracy, but more for their convenience, low risks, and low costs.
Essentially, these serology blood tests look for your antibody response to an infection in the blood stream. This is not direct detection of infection, but rather a measurement of how your immune system is responding with specific antibodies. If Lyme disease stayed in the blood stream, then serology tests would be nearly perfect. But Borrelia finds places to hide and ways to diminish the immune response.
The ELISA Test
The most common of the antibody tests is the Enzyme Linked Immune Sera Assay test (ELISA). This test is often the first test given to Lyme patients and can only give you a very general idea of the presence or absence of anti-Lyme antibodies.
There are many drawbacks to using the ELISA test for Lyme Disease:
• First, each lab can have their own version of an ELISA test, using different enzyme-linked antigens. For example none test uses a single protein only called the C6-Peptide protein. Other ELISA tests may use several bacterial proteins.
• Is more better? Generally yeas. But all ELISA tests have reliability issues.
Bacterial proteins are linked to an enzymatic reagent and are used to trap out the patient’s anti-Lyme antibodies from a serum sample.
When the antigen-linked-enzyme is in the presence of the correct anti-Lyme antibody, there is an enzymatic color change that occurs which is read by a machine. This is all done in what is called a dilution series, and is done in a small plastic plate that looks like a miniature clear plastic egg-carton with 50 Wells,
If the lab chooses bacterial antigens that do not reflect the same set of antibodies that the patient is producing, then the test will fail to detect Lyme disease. The individual that tests negative with one lab’s test may in fact have detectable Lyme antibodies if another lab’s ELISA test is given that employs a slightly different set of antigens.
Think of it like this: If you take a gold fish bowl and drop a paperclip and a penny into the bowl and then use a magnet to find the paper clip, you cannot conclude that there is no other metal in the bowl.
The magnet is simply incapable of detecting the penny. The same is true if you look for the wrong antibodies in a Lyme patient. The antibodies are there, but you’re simply unable to detect them.]
• Another problem is inconsistent accuracy within labs in interpreting results. Lori Bakken PhD Madison WI, did a survey of labs across the USA and showed the lack of consistency within laboratories to be able to faithfully reproduce the same result in an identical serum sample.
The study showed that more than 50% of the time labs could not correctly or consistently replicate identical results in identical triple-paired serum samples that were highly positive for Lyme antibodies.
This lack of accuracy is despite advertising claims by many labs of being 90+% specific and accurate.
However, accuracy by a laboratory’s definition has nothing to do with actual accuracy as a diagnostic test to determine the presence or absence of active infection in a patient.
• Bakken LL, Callister SM, Wand PJ, Schell RF. Interlaboratory Comparison of Test Results for the Detection of Lyme Disease by 516 Participants in the Wisconsin State Lab of Hygiene/College of American Pathologists Proficiency Testing Program. J Clin Microbiol 1997; Vol 35, No. 3:537-543
• Bakken LL, Case KL, Callister SM et al. Performance of 45 Laboratories Participating in a Proficiency Testing Program for Lyme Disease Serology. JAMA 1992;268:891-895
• Companies that are marketing their products often use buzzword terms like specific, sensitive, or accurate. These marketing terms do not mean diagnostically accurate! It usually refers to an internal reference test that the lab performs on their product under controlled conditions. For example if you perform 100 ELISA tests under controlled laboratory conditions, then you add a known amount of Lyme antibody, and 99 out of 100 showed a positive reaction, then you could say there was a 99% accuracy and a 1% false negative rate.
This does not mean you could take one hundred Lyme patients and give them the same exact test and expect the same results. Nor would it be correct to believe that the test is 99% accurate in determining if a patient is infected. But the way lab tests are marketed and promoted, this is exactly what many physicians believe! Since most doctors won’t treat Lyme without a positive test, they are excepting that the antibody tests are more accurate than diagnosing by a patient’s symptoms. A negative test is often the end of the line for a patient with seronegative Lyme.
What is a dilution Series?: ELISA tests are usually reported as a dilution series. If one part serum is added to one part distilled water it is a 1:1 dilution. The next dilution is 1:2 then 1:4 etc. Some labs won’t report a test positive unless they see an enzymatic color change at 1:64 or higher, but most labs use 1:256 as a cut off.
Extremely conservative labs use 1:1024 as a cut off which is so high that false negatives are common.
It is important for the doctor and patient to know what lab they are using and what the cut off point is in the dilution series to be considered positive. A simple positive or negative report without reporting the patient’s titer or referencing the cut off point is not acceptable. A sample that is positive at one lab at 1:256 is negative at the lab that reports out at 1:1024
Caveat emptor!
A lab may have a very sensitive and specific test but if the cut off that the lab uses is too high to reflect a patient’s average antibody response then the test is worthless in all but those patients who have an extremely high antibody response. So sensitivity becomes a meaningless term.
It would be almost criminal to tell a Lyme patient with an ELISA titer of 1:512 titer that he/she is negative simply because the lab has arbitrarily picked 1:1024 as the cutoff for positive tests. Any physician who wants to make a medically sound decision about treatment needs to see the patient’s titer and to consider the patient’s symptoms.
(ELISA: Enzyme-Linked Immunosorbent Serum Assay)
Barbour AG. Laboratory Aspects of Lyme borreliosis. Clin Microbiol Rev 1988;1:399-414
Cameron DJ. False Positive. [Letter/Commentary on Arch Internal Med 1991;151(9):1837-40] Arch Internal Med 1992;152(6):1331
Coyle PK. Borrelia burgdorferi specific antigen-antibody complexes in the CSF of 78 % of seronegative Lyme patients with early EM rashes. Fifth International Conference on Lyme Borreliosis, Arlington, VA, June 1992 and LDF Conference Atlantic City 1993.*
Coyle PK, Schutzer SE, Belman AL, Krupp LB, Golightly MG. Cerebrospinal fluid immune complexes in patients exposed to Borrelia burgdorferi: Detection of Borrelia-specific and non-specific complexes. Annal of Neurology, 1990;28:6:739-744
Craft JE, Grodzicki RL, Steere AC. Antibody response in Lyme Disease: an evaluation of diagnostic tests. J Infect Dis 1988 158:754-60
Craft JE, Fisher DK, et al. Antigens of Borrelia burgdorferi recognized during Lyme disease, Appearance of a new immunoglobulin M response and expansion of the IgG response late in the illness. J Clin Invest 1986;78:934-939
Dattwyler RJ, Volkman DJ et al. Seronegative Lyme Disease. New Eng J of Med 1988;319:1441-1446
Golightly MG. Laboratory considerations in the diagnosis and management of Lyme Borreliosis. [Review Article with 51 references] Amer J Clin Pathol 1993;99(2):168-74
Hansen K, Hinderson P, Pederson NS. Measurement of antibodies to Borrelia burgdorferi flagellum improves serodiagnosis in Lyme disease. J Clin Microbiol 1988;26:338-346
Hansen, Klaus. Serodiagnosis of erythema Migrans and Acrodermatitis Chronica Atrophicans by Borrelia burgdorferi flagellum ELISA. J Clin Microbiol, March 1989;27(3):545-551
Hedberg CW, Osterholm MT, MacDonald KL, White KE. An inter-laboratory study of antibody to Borrelia burgdorferi. J Infect Dis 1987;155:1325-27
Johnson BJB, Robbins KE, Bailey RE, et al. Serodiagnosis of Lyme Disease: Accuracy of a Two-Step Approach Using a Flagella-Based ELISA and Immunoblotting. J Infect Dis 1996;174:346-53
Kaiser R, Lucking CH. Intrathecal Synthesis of Specific Antibodies in Neuroborreliosis.
J Neurological Sciences, 1993;118:64-72
Magnarelli LA. Quality of Lyme disease tests. JAMA 1989;262:3464-3465
Magnarelli LA, Anderson JF. Enzyme-Linked Immunosorbent Assays for the Detection of Class-Specific Immunoglobulins to Borrelia burgdorferi.
American J of Epidemiology, April 1988;127(4)818-825
Magnarelli LA, Anderson JF, Johnson RC et al. Comparison of Different Strains of Borrelia burgdorferi Sensu Lato Used as Antigens in ELISA. J Clin Micro, May 1994;32(5):1154-1158
Magnarelli LA. Serologic Diagnosis of Lyme Disease. Annals New York Academy of Sciences, 1988;539:154-161
Magnarelli LA, Anderson JF, Johnson RC. Cross Reactivity in Serological Tests for Lyme Disease and other Spirochetal Infections. J Infectious Diseases, July 1987;156(1):133-138
Russell H, Sampson JS, Schmid GP, Wilkonson HW, Plikaytis B. Enzyme-linked immunosorbent assay for Lyme disease. J Infect Dis 1984;149:465-470
Schwartz BS, Goldstein MD, Rebeiro JMC, Schulze TL. Antibody testing in Lyme disease: A comparison of results in four different laboratories. JAMA. 1989;262:3431-3434
Sczepanski A, Benach JL. Lyme Borreliosis: Host response to Borrelia burgdorferi. Microbiol Review 1991;55:21
Weiss NL, Phillips MR, Sadock VA, Sigal LH, et al. False positive seroactivity to Borrelia burgdorferi in Rheumatic disease: The value of immunoblotting. Autoimmunity 1994 *
Weyand CM, Gorozny JJ. Immune responses to Borellia burgdorferi in patients with reactive Lyme arthritis. Arthritis Rheum. 1989;32:1057-1064
Zoller L, Haude M, Hassler D et al. Spontaneous and post-treatment antibody kinetics in late Lyme Borreliosis. Serodiagnosis Immunother Infect Dis 1989;3:345-353
Zoschke DC. Is it Lyme disease? How to interpret results of laboratory testing. Postgrad Med, 1992;91(7):46-55
Just as some Tick-Borne-Relapsing-Fever patients have reported bull’s-eye rashes, some Lyme patients have reported relapsing febrile states similar to Tick-Borne-Relapsing-Fever patients.
All Borrelia bacteria evolved from the same ancient ancestors. That means that all of the Borrelia species are linked by genetics. Ignoring those similarities has led to many unnecessary squabbles by clinicians over what defines “Lyme disease” as a separate disease, and how scientific semantics keeps us away from having discussions about Tick-Borne-Relapsing -Fever Borrelia species.
When talking about Lyme disease testing, why is it important to classify Lyme disease as part of a larger group of similar Borrelia pathogens?
The answer: All Borrelia infections are bad, so why not have an inclusive test that that includes all species of pathogenic Borrelia. I once commissioned Gene-Link to make an all inclusive PCR test for all Lyme species and pathogenic Relapsing Fever species. The cost would have been about $12,000
Any test that is specific for detecting B. burgdorferi, is mostly useless for new variations of Borrelia that cause Lyme-Like disease such as what is being seen in Missouri. Or the newly emerging species of Lyme disease like B.kurtenbachia or B. carolinensis and 12 others and more to come.
In Missouri most “Lyme-like-Disease” is caused by Borrelia lonestarri. The current Lyme tests are not as sensitive to B. lonestarri, but the symptoms and consequences are identical. While health departments and clinicians argue over names and definitions, patients are being misdiagnosed and ignored and worst of all told they don’t have true Lyme disease and are sent home with nothing but their symptoms.
Categories of Lyme Tests
There are five main methods of testing for Lyme disease: *
1. Antibody Tests: (The Elisa and Western Blot serology tests are most commonly used.)
2. PCR Tests: Bacterial DNA detection by polymerase-chain-reaction test (PCR)
3. Live culture of the bacteria from the body
4. Antigen Detection Tests: Finding particles and proteins of the bacteria in blood, CSF, urine, or tissue samples.
5. Direct observation by microscope: This can employ the use of biopsy and stain, or
centrifuged blood, spinal fluid, and urine.
*Other indirect tests such as T-cell activation test, Immune Fluorescence Tests, the Lyme Urine Antigen test, and the Immune Sera Culture Assay will not be explained in this booklet.
ANTIBODY TESTS
Antibody tests are by far the most commonly used tests for Lyme disease, and unfortunately one of the most misunderstood tests by physicians. These tests are indirect measurements of your body’s response to the bacterial pathogen.
Antibody serology tests are most often employed not for their accuracy, but more for their convenience, low risks, and low costs.
Essentially, these serology blood tests look for your antibody response to an infection in the blood stream. This is not direct detection of infection, but rather a measurement of how your immune system is responding with specific antibodies. If Lyme disease stayed in the blood stream, then serology tests would be nearly perfect. But Borrelia finds places to hide and ways to diminish the immune response.
The ELISA Test
The most common of the antibody tests is the Enzyme Linked Immune Sera Assay test (ELISA). This test is often the first test given to Lyme patients and can only give you a very general idea of the presence or absence of anti-Lyme antibodies.
There are many drawbacks to using the ELISA test for Lyme Disease:
• First, each lab can have their own version of an ELISA test, using different enzyme-linked antigens. For example none test uses a single protein only called the C6-Peptide protein. Other ELISA tests may use several bacterial proteins.
• Is more better? Generally yeas. But all ELISA tests have reliability issues.
Bacterial proteins are linked to an enzymatic reagent and are used to trap out the patient’s anti-Lyme antibodies from a serum sample.
When the antigen-linked-enzyme is in the presence of the correct anti-Lyme antibody, there is an enzymatic color change that occurs which is read by a machine. This is all done in what is called a dilution series, and is done in a small plastic plate that looks like a miniature clear plastic egg-carton with 50 Wells,
If the lab chooses bacterial antigens that do not reflect the same set of antibodies that the patient is producing, then the test will fail to detect Lyme disease. The individual that tests negative with one lab’s test may in fact have detectable Lyme antibodies if another lab’s ELISA test is given that employs a slightly different set of antigens.
Think of it like this: If you take a gold fish bowl and drop a paperclip and a penny into the bowl and then use a magnet to find the paper clip, you cannot conclude that there is no other metal in the bowl.
The magnet is simply incapable of detecting the penny. The same is true if you look for the wrong antibodies in a Lyme patient. The antibodies are there, but you’re simply unable to detect them.]
• Another problem is inconsistent accuracy within labs in interpreting results. Lori Bakken PhD Madison WI, did a survey of labs across the USA and showed the lack of consistency within laboratories to be able to faithfully reproduce the same result in an identical serum sample.
The study showed that more than 50% of the time labs could not correctly or consistently replicate identical results in identical triple-paired serum samples that were highly positive for Lyme antibodies.
This lack of accuracy is despite advertising claims by many labs of being 90+% specific and accurate.
However, accuracy by a laboratory’s definition has nothing to do with actual accuracy as a diagnostic test to determine the presence or absence of active infection in a patient.
• Bakken LL, Callister SM, Wand PJ, Schell RF. Interlaboratory Comparison of Test Results for the Detection of Lyme Disease by 516 Participants in the Wisconsin State Lab of Hygiene/College of American Pathologists Proficiency Testing Program. J Clin Microbiol 1997; Vol 35, No. 3:537-543
• Bakken LL, Case KL, Callister SM et al. Performance of 45 Laboratories Participating in a Proficiency Testing Program for Lyme Disease Serology. JAMA 1992;268:891-895
• Companies that are marketing their products often use buzzword terms like specific, sensitive, or accurate. These marketing terms do not mean diagnostically accurate! It usually refers to an internal reference test that the lab performs on their product under controlled conditions. For example if you perform 100 ELISA tests under controlled laboratory conditions, then you add a known amount of Lyme antibody, and 99 out of 100 showed a positive reaction, then you could say there was a 99% accuracy and a 1% false negative rate.
This does not mean you could take one hundred Lyme patients and give them the same exact test and expect the same results. Nor would it be correct to believe that the test is 99% accurate in determining if a patient is infected. But the way lab tests are marketed and promoted, this is exactly what many physicians believe! Since most doctors won’t treat Lyme without a positive test, they are excepting that the antibody tests are more accurate than diagnosing by a patient’s symptoms. A negative test is often the end of the line for a patient with seronegative Lyme.
What is a dilution Series?: ELISA tests are usually reported as a dilution series. If one part serum is added to one part distilled water it is a 1:1 dilution. The next dilution is 1:2 then 1:4 etc. Some labs won’t report a test positive unless they see an enzymatic color change at 1:64 or higher, but most labs use 1:256 as a cut off.
Extremely conservative labs use 1:1024 as a cut off which is so high that false negatives are common.
It is important for the doctor and patient to know what lab they are using and what the cut off point is in the dilution series to be considered positive. A simple positive or negative report without reporting the patient’s titer or referencing the cut off point is not acceptable. A sample that is positive at one lab at 1:256 is negative at the lab that reports out at 1:1024
Caveat emptor!
A lab may have a very sensitive and specific test but if the cut off that the lab uses is too high to reflect a patient’s average antibody response then the test is worthless in all but those patients who have an extremely high antibody response. So sensitivity becomes a meaningless term.
It would be almost criminal to tell a Lyme patient with an ELISA titer of 1:512 titer that he/she is negative simply because the lab has arbitrarily picked 1:1024 as the cutoff for positive tests. Any physician who wants to make a medically sound decision about treatment needs to see the patient’s titer and to consider the patient’s symptoms.
(ELISA: Enzyme-Linked Immunosorbent Serum Assay)
Barbour AG. Laboratory Aspects of Lyme borreliosis. Clin Microbiol Rev 1988;1:399-414
Cameron DJ. False Positive. [Letter/Commentary on Arch Internal Med 1991;151(9):1837-40] Arch Internal Med 1992;152(6):1331
Coyle PK. Borrelia burgdorferi specific antigen-antibody complexes in the CSF of 78 % of seronegative Lyme patients with early EM rashes. Fifth International Conference on Lyme Borreliosis, Arlington, VA, June 1992 and LDF Conference Atlantic City 1993.*
Coyle PK, Schutzer SE, Belman AL, Krupp LB, Golightly MG. Cerebrospinal fluid immune complexes in patients exposed to Borrelia burgdorferi: Detection of Borrelia-specific and non-specific complexes. Annal of Neurology, 1990;28:6:739-744
Craft JE, Grodzicki RL, Steere AC. Antibody response in Lyme Disease: an evaluation of diagnostic tests. J Infect Dis 1988 158:754-60
Craft JE, Fisher DK, et al. Antigens of Borrelia burgdorferi recognized during Lyme disease, Appearance of a new immunoglobulin M response and expansion of the IgG response late in the illness. J Clin Invest 1986;78:934-939
Dattwyler RJ, Volkman DJ et al. Seronegative Lyme Disease. New Eng J of Med 1988;319:1441-1446
Golightly MG. Laboratory considerations in the diagnosis and management of Lyme Borreliosis. [Review Article with 51 references] Amer J Clin Pathol 1993;99(2):168-74
Hansen K, Hinderson P, Pederson NS. Measurement of antibodies to Borrelia burgdorferi flagellum improves serodiagnosis in Lyme disease. J Clin Microbiol 1988;26:338-346
Hansen, Klaus. Serodiagnosis of erythema Migrans and Acrodermatitis Chronica Atrophicans by Borrelia burgdorferi flagellum ELISA. J Clin Microbiol, March 1989;27(3):545-551
Hedberg CW, Osterholm MT, MacDonald KL, White KE. An inter-laboratory study of antibody to Borrelia burgdorferi. J Infect Dis 1987;155:1325-27
Johnson BJB, Robbins KE, Bailey RE, et al. Serodiagnosis of Lyme Disease: Accuracy of a Two-Step Approach Using a Flagella-Based ELISA and Immunoblotting. J Infect Dis 1996;174:346-53
Kaiser R, Lucking CH. Intrathecal Synthesis of Specific Antibodies in Neuroborreliosis.
J Neurological Sciences, 1993;118:64-72
Magnarelli LA. Quality of Lyme disease tests. JAMA 1989;262:3464-3465
Magnarelli LA, Anderson JF. Enzyme-Linked Immunosorbent Assays for the Detection of Class-Specific Immunoglobulins to Borrelia burgdorferi.
American J of Epidemiology, April 1988;127(4)818-825
Magnarelli LA, Anderson JF, Johnson RC et al. Comparison of Different Strains of Borrelia burgdorferi Sensu Lato Used as Antigens in ELISA. J Clin Micro, May 1994;32(5):1154-1158
Magnarelli LA. Serologic Diagnosis of Lyme Disease. Annals New York Academy of Sciences, 1988;539:154-161
Magnarelli LA, Anderson JF, Johnson RC. Cross Reactivity in Serological Tests for Lyme Disease and other Spirochetal Infections. J Infectious Diseases, July 1987;156(1):133-138
Russell H, Sampson JS, Schmid GP, Wilkonson HW, Plikaytis B. Enzyme-linked immunosorbent assay for Lyme disease. J Infect Dis 1984;149:465-470
Schwartz BS, Goldstein MD, Rebeiro JMC, Schulze TL. Antibody testing in Lyme disease: A comparison of results in four different laboratories. JAMA. 1989;262:3431-3434
Sczepanski A, Benach JL. Lyme Borreliosis: Host response to Borrelia burgdorferi. Microbiol Review 1991;55:21
Weiss NL, Phillips MR, Sadock VA, Sigal LH, et al. False positive seroactivity to Borrelia burgdorferi in Rheumatic disease: The value of immunoblotting. Autoimmunity 1994 *
Weyand CM, Gorozny JJ. Immune responses to Borellia burgdorferi in patients with reactive Lyme arthritis. Arthritis Rheum. 1989;32:1057-1064
Zoller L, Haude M, Hassler D et al. Spontaneous and post-treatment antibody kinetics in late Lyme Borreliosis. Serodiagnosis Immunother Infect Dis 1989;3:345-353
Zoschke DC. Is it Lyme disease? How to interpret results of laboratory testing. Postgrad Med, 1992;91(7):46-55
