Interpreting western blots for Lyme attn Tim

[Guest guest]

I am posting here an excellent and simple, though long, summary by

Dr. Crist on how to interpret the IgeneX western blot. You could

interpret any lab's western blot for borrelia this way IF YOU COULD

GET THEM TO REPORT ON ALL THE BANDS. Nuff said. Here is the report.

It is invaluable and work printing, reading and saving. I have not

seen this addressed any better by anybody. (a Carnes)

Posting this, written by Dr C of Missouri for the benefit of

everyone. This was written around 1999 or 2000. There is an updated

version below.

Please note that " equivocal " is the same thing as " IND "

or " indeterminate. "

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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 Donta, M.D. lectured.

Dr. Donta 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. Horowitz, 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

.

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 Mc, Ph.D. has shown that there is a different

borrelia in the Midwestern U.S.A. When Dr. Mc 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. Mc 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.

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.

Testing in my office consists of a Western blot that is express-

mailed to a borreliosis reference laboratory.