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Lyme Community › Lyme Articles › Dr C's Western Blot Explanation Lyme Facts
|Dr C's Western Blot Explanation Lyme Facts|
|Written by Canuck|
|28 February 2009|
Please note that "equivocal" is the same thing as "IND" or "indeterminate."
Explaining Borreliosis (Lyme) Western Blot Tests
The Western blot is a type of test that is conducted for detection of borreliosis (Lyme), but is also used to test for infections other than borreliosis.
Borreliosis is a more accurate name than Lyme disease for this infection. Several different Borrelia may cause a similar clinical pattern in this disease.
Old Lyme is a town in Connecticut, not a disease. Borreliosis is the name that should be used.
There is no universal agreement on what defines a positive Western blot.
Good laboratories use different criteria to interpret borreliosis blots. At the 1999 international borreliosis and tick-borne infection conference, Sam D****, M.D. lectured.Testing in my office consists of a Western blot that is express-mailed to a borreliosis reference laboratory.
Dr. D**** is a full professor of Infectious Disease at Boston University School of Medicine. He said that if a patient has just one borreliosis-associated antibody on their Western blot, you may assume they have borreliosis. Richard H*****, M.D. said the same thing in his lecture, at that same conference.
Research I presented in 1998 involving over 400 borreliosis patients, showed an 87% response rate to antibiotics. This was if they had one borreliosis-associated antibody on their blot.
So if there is enough suspicion that Lyme borreliosis is the cause of a patient's symptoms, so much so that a Western blot is ordered, then if only one borreliosis-associated antibody is found, it is significant!
Medical literature is replete with statements about false positive test results for Lyme borreliosis. Since 1988, I have diagnosed and treated well over 600 borreliosis patients. Only 2 of those patients with a positive borreliosis test did not respond to antibiotics. This is a 99% success rate!
So in the trenches of day-to-day medical practice, false positive borreliosis tests are not an issue. In retrospect, those 2 patients that did not respond to antibiotics may have also had babesiosis.
In my practice, many borreliosis patients also have babesiosis, another tick-borne infection that causes the same symptoms as Lyme borreliosis.
Babesiosis is caused by a protozoa, which is a different germ type than a bacteria, virus, fungus or yeast.
The placebo effect would not explain a 99% response rate. Those borreliosis associated antibodies should not be there, in patients with symptoms.
A placebo is like a sugar pill, that has no effect. A placebo effect occurs because patients believe in the pill they are taking, even though it is a sugar pill. The human mind causes the response. Placebo effects should more likely be about 20-30%, not a 99% response rate.
False negative test results are the real problem in diagnosing borreliosis. Research has shown that you have to do the right test (the Western blot), done at the right laboratory (one that specializes in testing borreliosis), and done the correct way (shipped express delivery early in the week).
The right test to screen for borreliosis is the Western blot. Research I presented in Bologna, Italy in 1994 at the international borreliosis conference showed this.
Other screening tests, such as the IFA, EIA, ELISA, and PCR DNA probe were often negative when the Western Blot was positive!
Other doctors like myself who diagnose and treat a lot of borreliosis patients, go straight to the Western blot as their screening test.
Medical articles abound stating that it is best to do a screening test, such as an ELISA, and if it is positive, then confirm it with a Western blot.
But the ELISA is often negative when the Western blot is positive so, the right test is the Western blot.
It lets you see exactly which antibodies are present. The "right laboratory" means one that specializes in borreliosis testing.
In the past, I have done head to head comparisons with 3 different regular labs. Western blots were drawn and sent on the same day to 2 different labs.
The labs that specialize in borreliosis testing typically found borrelia-associated antibodies, that the regular laboratories missed.
If these specialty labs find a borrelia antibody, I trust it to be significant, because patients respond to antibiotics.
You get what you pay for, so use a lab that specializes in borreliosis. The right way to process the Western blot specimen means for the blood to be drawn and express mailed early in the week.
Research shows the borrelia antibodies have the potential to clump together, resulting in false negative test results. So far, unclumping has not been practical for laboratories to do.
The fresher the specimen, the more accurate the test results. Patients at our office are scheduled Monday, Tuesday, or Wednesday if testing is to be done.
This way, express shipping will assure that the specimen does not spend the weekend sitting at the post office. This is the right way to test and ship borreliosis specimens.
Western blots look for antibodies. These antibodies are made by your immune system. In this case, the antibodies are made to fight against different parts of the Lyme bacteria, which is called Borrelia burgdorferi, and other Borrelia species.
In other words, your immune system does not make one big antibody against the whole bacteria. So, when you see a number on a borreliosis Western blot, it corresponds to a specific part of the bacteria.
Compare it to the old story of different blind people touching an elephant. Based on the part of the elephant each one touched, each person had their own perception. Likewise, the antibodies attach to different and specific parts of Borrelia burgdorferi.
Numbers on Western blots correspond to weights. Kilodaltons (kDa) are the units used for these microscopic weights. Think of it like pounds or ounces. An 18 kDa antibody weighs 18 kilodaltons.
To do a Western blot, thin gel strips are impregnated with the various parts of Borrelia burgdorferi. Each of the numbers, 18 through 93, on the test result form, is a part of the bacteria.
Blood is made up of red blood cells and serum; Spinning blood in a centrifuge separates serum from red blood cells and other things, like white blood cells and platelets.
Serum contains antibodies made by the immune system. Electricity is used to push the serum through the thin gel strips for the Western blot.
If there are any antibodies against parts of Borrelia burgdorferi present in your serum, and these parts are impregnated on the strip, the antibody will complex (bind) to that part.
When antibodies form a complex, it is called an antigen-antibody complex. Anything foreign in the body is an antigen, such as a ragweed pollen particle, germ, cancer, and even a splinter.
In the case of borreliosis, the various parts of Borrelia burgdorferi are all antigens. Though each antigen is different, they all come from the same bacteria. So all the numbers that are positive on the test report are due to antigen-antibody complexes.
If enough of the complexes are formed, eventually it may be seen with the naked eye as a dark band. - Band intensity reflects how dark or wide it is. Controversy exists about band intensity.
Many would say the " +/-" equivocal ["IND"] bands are not significant. The problem I have with that, is that there are "-" negative bands. The lab has no trouble calling some bands negative. So they must be seeing something when they put "+/-" at some bands.
The only thing that makes sense, is that there is a little bit of that antibody present in your serum. If the "+/-" equivocal is reported on the borrelia associated bands, it is usually significant, in my clinical experience. This is a strong clue that I am on the right track.
Instead of ignoring these, they should be a red flag to keep pursuing a laboratory diagnosis. Giving patients 4 weeks of antibiotics (usually tetracycline, 500 mg, 3 times a day), will convert a negative or equivocal Western blot to positive in about 36% of cases.
As mentioned, if these positive blots are found by specialty labs, over 99% of those patients will respond to antibiotics.
Sometimes multiple antibiotics have to be tried before the patient feels better. Antibiotics may actually help with the laboratory diagnosis. But patients need to be off antibiotics about 10 to 14 days before the Western blot is repeated. This sounds like a contradiction.
Antibiotics may help convert the test to positive, but patients need to be off antibiotics when the specimen is drawn.
It is well documented in medical literature that the presence of antibiotics may cause false negative borreliosis testing. Therefore, your system should be free of all antibiotics for an accurate blot result.
When the Lyme borrelia are alive, they are geniuses at avoiding the immune system. They may do things like go inside your white blood cells, and come out enclosed by the cell membrane of your own white blood cells! This may partly explain why antibodies against Borrelia burgdorferi are often not found when patients are tested.
What may happen when patients are given 4 weeks of tetracycline (or other antibiotics) is that some of the bacteria die. When Borrelia burgdorferi dies, it is less efficient at avoiding the immune system.
That's when antibodies may be formed against Borrelia burgdorferi, converting the negative or equivocal Western blot to positive, in about 36% of cases.
If a borreliosis Western blot is going to be positive, it is usually the first one that is positive. The second blot is the next most likely to be positive, and so on, until the fifth blot.
After that, the curve levels off for conversion to positive. This is based on research I presented in Bologna, Italy in 1994. Some patients had borrelia-associated antibodies finally show on their tenth Western blot! Two Western blots from a reliable lab usually gives the answer.
If a third test is needed, a Lyme Urine Antigen Test (LUAT) is done instead of a third Western blot. Positive LUATs correspond very highly to patients getting better with antibiotics.
False positive LUATs have not been a problem in my practice. The LUAT finds the actual antigen (Borrelia burgdorferi itself), so arguably it should be the test of choice, but the Western blot is rn6re widely accepted, even though it looks for the antibodies against Borrelia burgdorferi.
The presence of antibodies are indirect evidence of an infection, not direct evidence like shown in the LUAT. On the Western blot test result form, please note what is "considered positive" and "considered equivocal." Equivocal is another way of saying suspicious or almost positive.
Below this are the ASTPHLD/CDC recommendations. The CDC stands for the Center for Disease Control. I have been in attendance at the international borreliosis conferences when the CDC said their recommendations are for disease surveillance, not day-to-day clinical medical practice. I am not in the business of disease surveillance. My job is to try to help sick people.
The CDC recommendations do not include the 31 and 34 Kda bands of the blot test. These two bands correspond to outer surface proteins A and B respectively (ospA and ospB).
In the world of borreliosis, these are two of the classic hallmark Lyme antibodies. But the CDC does not even have them in their recommendations.
You may see why I and other borreliosis clinicians do not agree with using the CDC criteria in everyday medical practice. Other bacteria besides Borrelia burgdorferi may produce the 45, 58, 66, and 73 kDa bands.
These bands may be produced by Borrelia burgdorferi, but are not nearly as specifically associated with Lyme borreliosis as the starred bands. These starred bands are classic hallmark borrelia-associated antigen-antibody complexes.
An example of the CDC's criteria of a blot test, is if a patient has the band pattern of 41, 45, 58, 66, and 93, the CDC would call it positive. But if a patient has a 23-25, 31, 34, and 39 band pattern, they would call it negative.
This is despite the fact that this second pattern of antigen-antibody complex bands is much more specifically associated with Borrelia burgdorferi than the first pattern.
As you can see, borreliosis is very controversial. It would be alarming if I was the only clinician who thought that the CDC recommendations should not be used for day-to day medical practice.
Many borrelia clinicians do not use the CDC criteria. This is obvious by the fact that the IgX laboratory uses different criteria for positive. Again, in my opinion and others', even one borrelia-associated antibody is significant, if symptoms exist.
The classic triad of symptoms for borreliosis is fatigue (tiredness, exhaustion), musculoskeletal pain (joints, muscles, back, neck, headache), and cognitive problems (memory loss, trouble concentrating, difficulty remembering what you read, depression, disorientation, getting lost).
But there are about 100 symptoms on the borreliosis questionnaire I use. Borreliosis may mimic or imitate virtually any disease.
Patients often tell me that other physicians they have seen use the CDC recommendations. This is unfortunate, in my opinion, since these physicians are not in the business of disease surveillance, like the CDC is.
But I am biased. After seeing patients with borreliosis since 1988, attending many conferences, talking with experts, and doing research on borreliosis testing, there is absolutely no question in my mind that physicians need to not blindly accept any recommendations.
One of my hopes is that doctors will someday realize that this controversy is a signal for them to search for the truth. Why is there such conflict in this very "political" disease if there is not substance for disagreement? Both IgG and IgM Western blots should be done for borreliosis.
With most infections, your immune system first forms IgM antibodies, then in about 2 to 4 weeks, you see IgG antibodies. In some infections, IgG antibodies may be detectable for years.
Because Borrelia burgdorferi is a chronic persistent infection that may last for decades, you would think patients with chronic symptoms would have positive IgG Western blots.
But actually, more IgM blots are positive in chronic borreliosis than IgG. Every time Borrelia burgdorferi reproduces itself, it may stimulate the immune system to form new IgM antibodies.
Some patients have both IgG and IgM blots positive. But if either the IgG or IgM blot is positive, overall it is a positive result.
Response to antibiotics is the same if either is positive, or both. Some antibodies against the borrelia are given more significance if they are IgG versus IgM, or vice versa.
Since this is a chronic persistent infection, this does not make a lot of sense to me. A newly formed Borrelia burgdorferi should have the same antigen parts as the previous bacteria that produced it.
But anyway, from my clinical experience, these borrelia associated bands usually predict a clinical change in symptoms with antibiotics, regardless of whether they are IgG or IgM. In regard to the outer surface proteins, think of it like the skin of a human.
On the outer surface of the Lyme bacteria are various proteins. As they have been discovered, they have been assigned letters, such as outer surface proteins A, B, and C.
The following is a brief explanation of the test results. Again, each band is an antigen complexed (bound together) with an antibody made by the immune system, specifically for that antigen (part) of Borrelia burgdorferi.
18: An outer surface protein.
22: Possibly a variant of outer surface protein C.
23-25: Outer surface protein C (osp C).
28: An outer surface protein.
30: Possibly a variant of outer surface protein A.
31: Outer surface protein A (osp A). 34: Outer surface protein B (osp B).
37: Unknown, but it is in the medical literature that it is a borrelia-associated antibody. Other labs consider it significant.
39: Unknown what this antigen is, but based on research at the National Institute of Health (NIH), other Borrelia (such as Borrelia recurrentis that causes relapsing fever), do not even have the genetics to code for the 39 kDa antigen, much less produce it. It is the most specific antibody for borreliosis of all.
41: Flagella or tail. This is how Borrelia burgdorferi moves around, by moving the flagella. Many bacteria have flagella. This is the most common borreliosis antibody.
45: Heat shock protein. This helps the bacteria survive fever. The only bacteria in the world that does not have heat shock proteins is Treponema pallidum, the cause of syphilis.
58: Heat shock protein.
66: Heat shock protein. This is the second most common borrelia antibody.
73: Heat shock protein.
83: This is the DNA or genetic material of Borrelia burgdorferi. It is the same thing as the 93, based upon the medical literature. But laboratories vary in assigning significance to the 83 versus the 93.
93: The DNA or genetic material of Borrelia burgdorferi.
In my clinical experience, if a patient has symptoms suspicious for borreliosis, and has one or more of the following bands, there is a very high probability the patient has borreliosis.
These bands are 18, 22, 23-25, 28, 30, 31, 34, 37, 39, 41, 83, and 93.
This is true regardless of whether it is IgG or IgM.. But again, there is no universal agreement on the significance of these bands. Betina Wilska, M.D. from Germany is one of the world's experts on outer surface protein A (31 kDa).
At the international borreliosis conference in Vancouver, British Columbia, I asked her personally about the 30 kDa band. She told me it was the same as the 31 kDa band (osp A).
When you have the opportunity to talk to borreliosis experts, this helps in assigning significance to findings, on an imperfect test. As a medical doctor, I am stating all of this with no axe to grind, no professorship to protect, and no preset opinions. Patients, personal research, and conferences have helped me interpret the borreliosis medical literature in regard to testing.
Nobody would like to have available a bullet-proof, 100% reliable Lyme borreliosis test more than I would. But we must use what is currently available. I always welcome second opinions.
Here is his update written sometime around 2005.
When physicians do consider borreliosis, they often start with a screening test such as an EIA, ELISA, IFA or PCR-DNA probe. If the initial screening test is negative, many physicians tell patients they do not have Lyme borreliosis and the testing is stopped right there.
Screening tests that are positive are often followed by a test called the Western blot. The blot is a “confirmatory” test, as opposed to a screening test.
(Blots are performed for other infection -- it is a type of test, not a test uniquely for the Lyme bacteria.)
Western blots are accomplished by breaking the Borrelia burgdorferi into pieces, and those parts of the Lyme bacteria are then embedded in a gel.
Electricity is used to push antibodies made by the immune system through the gel. Antibodies that are made to attach to certain parts of the Lyme bacteria will bind to those exact parts that are embedded in the gel.
When the antibodies bind to the parts of the bacteria, a black band is formed, which is then interpreted as +/-, +, ++ or +++ depending upon the intensity or darkness of the band.
Each part of the Lyme bacteria weighs a certain amount. For example, the tail of the Lyme bacteria weighs 41 kilodaltons (kDa).
Think of kilodaltons like pounds, ounces or kilograms. The numbers on a Western blot such as 23, 31, 34 or 39 refer to how much that particular part of the bacteria weighs in kilodaltons.
The significant antibodies, in my opinion, are the 18, 23-25, 28, 30, 31, 34, 39, 58, 66 and 93.
It’s important to know that screening tests like the EIA, ELISA, IFA and PCR can be negative even when the Western blot (confirmatory test) is positive.
I presented research that supported this at the 1994 International Lyme Borreliosis Conference held in Bologna, Italy.
For this reason I believe the screening tests are practically worthless, and is why I use the Western blot to “screen” for borreliosis, even though it is a “confirmatory” test.
Antibodies are very specific as to what they bind; consequently, in over 700 borreliosis patients false positive blot results occurred in only three percent of them, based upon research I presented at the 2000 International Lyme Borreliosis conference.
Data from those same 700 patients showed that if their Western blots had even one antibody significantly associated with the Lyme bacteria, then there was a 97 percent chance they would feel better with antibiotics.
Consequently, I tell my patients not to worry if the laboratory interpretation is “negative” or “equivocal,” if they have antibodies that are significantly associated with Borrelia burgdorferi.
One thing doctors are taught in medical schools is to treat the patient, not the test result.
If someone has chronic pain, fatigue, cognitive problems, blurry vision and/or neurological problems, and also has a significant antibody on a borreliosis Western blot, that antibody should not be ignored in my opinion, even if the ‘official’ interpretation is negative or equivocal.
Remember, antibodies are very specific to what they bind, and borreliosis may cause virtually any symptom and any disease.
Disease surveillance is close observation of a group of patients with the same disease, and it is one of the jobs of the Centers for Disease Control (CDC).
Criteria used for disease surveillance is often different than criteria used to diagnose and treat patients. In my opinion, surveillance criteria should not be used in day-to-day clinical medical practice.
Unfortunately, many patients are told they do not have borreliosis because they do not meet CDC’s surveillance criteria.
Surveillance criteria exclude some of the classic hallmark antibodies, such as the 31 kDa band (outer surface protein A or ospA) and the 34 kDa band (outer surface protein B or ospB).
In fact, the 31 kDa band is so tightly associated with Lyme borreliosis that a vaccine was made from that outer surface protein.
In other words, I believe that criteria that exclude the ospA (31 kDa) band should not be used to tell a patient they do not have Lyme borreliosis.
Common sense should tell anyone that prevalent antibodies like the 31 dKa and 34 dKa should be included in the criteria, not excluded.
(Remember, research supports that if just one antibody that is significantly associated with Borrelia burgdorferi is present on a Western blot, 97 percent of those patients with chronic symptoms or chronic diseases feel better with antibiotics.)
Same day head-to-head comparisons of borreliosis Western blot results revealed that reference laboratories do a better job of finding antibodies against Borrelia burgdorferi than regular laboratories.
This raised the obvious concern that the reference labs might be overdiagnosing patients with borreliosis.
That is one of the reasons why I researched those 700 patients. However, the false positive rate was just three percent. In my opinion, reference laboratories do not over-diagnose borreliosis.
False negative test results, on the other hand, are a much bigger problem, in my experience. Negative Western blots convert to positive in 18 to 24 percent of cases, if four weeks of antibiotics are given, and then the patients go off antibiotics for 10 to 14 days before the repeat Western blots are done.
In other words, a false negative Western blot converts to positive in about one out of five borreliosis patients. This is a much greater problem than a false positive rate of only three percent.
Coinfection testing may depend upon where you live on planet earth. I talked to one medical doctor from New England that was concerned about getting too many positive test results for bartonellosis (cat scratch disease).
This physician was concerned about false positives. Yet I have not had a single positive yet.
Research by Greg McDonald, Ph.D. has shown that there is a different borrelia in the Midwestern U.S.A. When Dr. McDonald used a PCR primer that would amplify any strain of borrelia, he obtained positives from biopsies of bulls-eye rashes caused by tick bites in patients from Missouri and nearby states.
However, if Dr. McDonald narrowed the PCR primers to amplify only Borrelia burgdorferi, Borrelia lonestari or Borrelia andersoni, the results were negative.
In other words, the Midwest has a different borrelia. It has been referred to as Borrelia “confusiosis,” but one of these years when it is finally characterized fully, this Midwestern borrelia will probably be known as Borrelia mastersi, in honor of Edwin Jordan Masters, M.D. and his extensive research.
Pathologists who use a microscope to examine bulls-eye rash biopsy specimens from Midwestern patients observe significant and consistent differences when compared to biopsies from New England patients.
The diseases and their rashes are similar, but there are definite differences. This is why borreliosis or Master’s disease is a better term than Lyme disease.
Another feature of Midwestern borreliosis is the inability to grow Borrelia burgdorferi from patients with Lyme borreliosis. In New England about five percent of cultures grow Borrelia burgdorferi from borreliosis patients.
There are other borrelia* that cannot be grown in culture media. The bacteria that causes syphilis has never been grown in culture media, even though this infection has been known and studied for several generations.
It should not be surprising that the Midwestern borrelia cannot be grown in culture media yet. When it is, knowledge of this infection will increase tremendously.
James Oliver, PhD, who is a very highly respected entomologist, has successfully cultured Borrelia burgdorferi from over 60 ticks collected in Missouri.
Why human cultures are negative and tick cultures are positive remains a mystery. Still, there is no question but that there is a Midwestern borreliosis.
The same is true for co-infections. The babesia in Missouri is called MO-1. It is a different babesia. There are different ehrlichia.
It would appear there is a different bartonella. When you have different strains of germs, the test results may be falsely negative.
To protect patients’ pocketbooks, I rarely test for tick-borne coinfections. If the tests were reliable I would be more inclined to order more. In general, when potential coinfections are targeted with antibiotics, most patients get better.
At least three possibilities exist to explain patients feeling better with antibiotics. It could be that an antibiotic that targets a potential coinfection such as babesiosis may actually be killing the Lyme bacteria as well.
Or it may be that a negative test for a coinfection was falsely negative. And finally, there may be some unknown germ that the patient has that responds to the antibiotic.
I tell my patients that regardless of why the antibiotics help most borreliosis patients, the benefits of antibiotics outweigh the risks.
My greatest concern is untreated borreliosis, not the potential side effects of antibiotics that target tick-borne infections.
Specimens for borreliosis Western blot testing should always be express-mailed to the laboratory. Antibodies against the Lyme bacteria can clump or bind together and give a false negative test result.
Express-mailing specimens lessens the time in which this could happen, which in turn increases test accuracy.
If your specimen sits around for several days (or if a screening test is ordered instead of a Western blot, or if a regular lab is used instead of a reference lab) then you might be given a false negative test result, which in turn could result in a false sense of security.
thanks to Bettyg for this one:)
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