Wednesday, December 27, 2017

All I got was a 41 band. All I got were IgM bands -- could it be chronic Lyme? The Western Blot revisited.

Many patients and others continue to inquire:

All I got was a 41 band - do I have Lyme?    Lyme test is IgM positive, could it be chronic Lyme?

Its complicated.

If the text is too hard to follow or boring, there are pictures below. 

It helps if you know a little about laboratory medicine.

The Western Blot and ELISA tests, the tests most often used, are indirect tests. Rather than looking for direct evidence of infection, e.g. finding specific DNA, These tests look for foot prints: Antibodies or specific immunoglobulins.  These tiny proteins are made by the immune system in response to recognized pathogenic (disease-causing) germs -- bacteria, fungi, protozoans and viruses primarily.
There are a lot of reasons why Lyme antibody tests are not that great:  People are genetically programmed to respond differently; Lyme strains vary, species vary; the commonly used test is based on a single Lyme strain and other species may not react to the test -- and some variations in immune response may be random.  Perhaps most important: the test is incorrectly calibrated, based on false assertions. 
Before we get to the Western Blot, let's first discuss the ELISA. Enzyme Linked Immunosorbent assay.  This is the test most commonly used, at least for screening. 
All diagnostic serological (from blood-serum) lab tests need controls, positive and negative.  
A positive control is a known "serum" full of Lyme antibodies, against which a patient's serum is compared. A negative control is a known serum, ostensibly with no Lyme antibodies. (The availability of true negative controls has been a debatable point).

In order to proceed, we need Lyme antigen -- proteins and polysaccharides, which stimulate antibody formation.  The raw material comes from a culture of a subspecies subset of Lyme bacteria. This is the stuff we make Lyme antibodies against if we are infected. 
Roughly, a bunch of Lyme bacteria are put in a "blender," or sonicated. 
ELISA.   Sticky Lyme antigen is adhered to the surface of small wells . Serum is added. Lyme antibodies present in the patient test serum should tightly bind to the Lyme antigens on the bottom of the well. There is some nonspecific binding by "promiscuous" antibodies that apparently will stick to anything. The test well are washed thoroughly to remove debris and nonspecific antibodies. After the first stage we have -- Lyme bacteria antigens "glued" to the bottom of a well (test tube equivalent) with Lyme antibodies tightly adhering to antigenic parts of the Lyme bacteria.  Antigenic parts are those that stimulate the immune system and thereby stimulate the production of antibodies. 
ELISA stands for Enzyme linked Immuno-sorbent assay. Antibodies are shaped like a Y.  There are 3 points of contact, with the potential to bind to a receptor  A second, known antibody which reacts to other antibodies -- this antibody grabs on to other antibodies, latches on to one of the remaining 3 legs of the Y shaped antigen bound antibody.  A protein called an enzyme is attached to a leg of the second antibody.  The enzyme turns color when a reagent is added to the tubes/wells. The intensity of color increases if more antibodies are bound to the antigen. Patient serum with a high concentration of Lyme antibodies induces an intense color change. If no Lyme antibodies are present no color change or only a slight color change occurs. A mild amount of color is thought to be normal and due to nonspecific, promiscuous antibodies bound to the Lyme antigens.  Basically:  More color, more antibodies; less color, fewer antibodies. The results are recorded as an index.

This test has the advantage of including all Lyme antibodies that might be present in the patient's serum. The test incorporates polysaccharide (sugar) based antigens and the Western Blot considers only protein antigens.

The test and the results it produces are controversial because of two differing beliefs about the inherent nature of the disease. One group thinks the test is inaccurate because it causes too many false positives.  The other group feels the test fails because of false negatives.

The test needs a cut off point -- how much color change is needed for a positive. The cut off for a positive test is based on a control.

The positive control is based on an ideal serum with a robust immune response, typically found in acute phase of the disease.  Excluded are: patients who have been sick for a long time, patients who have a weak immune response, patients with a different strain of Lyme and many others. 
To make matters worse, the test is entirely predicated on a single strain of Borrelia burgdorferi, the B31 strain. The test will like fail when a patient is infected with other strains and/or species of Lyme.

Doctors talk about whether or not a test is sensitive or it is specific.

A test is sensitive if it excludes false negatives. A test is specific when all the positive are "true positives." There is always a balance. When you increase sensitivity you decrease specificity. When you increase specificity you decrease sensitivity.  
The ELISA I state of the art technology and widely used. Why does Lyme testing require 2 steps, an ELISA followed by a Western Blot?  In the past the same procedure was needed for HIV. Now, with a new and improved ELISA, an HIV Western blot is no longer required. The complexity of testing underlies the overarching war about the disease in general.

The Western Blot is considered both a sensitive and specific test. It is superior to the ELISA. A mix of Lyme antigens is placed into a gel.  When an electric current is applied, proteins from the Lyme parts separate out based on the weight of individual proteins. Antigenic proteins are spread out in a linear fashion across the gel.  The separated proteins are transferred to a strip of nitrocellulose -- something like a strip of paper. The test proteins typically was molecular weights of 18 kilo Daltons to 93 kilo Daltons. 
The antigen/protein laden strips are incubated with serum. Antibodies present in the serum will bind to the individual proteins on the strips. A reagent is added and bars or bands appear across the strip.
Band position and name corresponds to the molecular weight of the particular Lyme antigen.
Some bands are very specific. The likelihood of non specific antibody binding to the teased out protein approximates is low.  The CDC formula is based on a total number of bands appearing from a set and does not consider the importance of particular bands. The CDC standard is derived from a 1994 convention at which time a standard for surveillance was established.

The Western Blot suffers with many of the shortcomings of the ELISA.  The accuracy of the test depends on the validity of the controls.

A picture is worth a thousand words. Let me a review a few images, typical of what I work with on a daily basis.




These results are from MDL.  A specialty laboratory in NJ.  This is an IgG strip. It incorporates a test for antibodies against 12 bands or 12 antigen proteins of the corresponding weight. Ten bands are based on the 1994 standard and 2 additional bands, 31 and 34 are tested separately. That is why these numbered bands are out of place on the strip.  IgM strips are prepared separately by using separate reagents.  The bottom strip is the positive control - high levels of specific antibody directed against the blot proteins. The top line is the patient result. No negative control is provided. 

CAVEAT;   INTERPRETATIONS DISCUSSED ARE THE OPINION OF THE AUTHOR AND MAY BE CONTROVERSIAL AND OPPOSED BY MOST AUTHORITIES. 

This test is CDC negative.
Patient antibodies react to nearly every band. This strip appears blatantly positive.  However, according to the manufacturer, the intensity of the response is too weak to call positive. A band is considered positive if the intensity is at least 60%  of the control. For example, the 93 band has an intensity of 45% of the control. Based on this standard, only the 41 band is counted positive. This interpretation is wrong, I think, because the lower limit of a positive results is incorrect -- based on incorrect assumptions regarding the control.  I call this test positive.   Any band 30% of the control or more -- and more so bands of 40% or more, likely represents Lyme antibody binding and not nonspecific throw away binding. 







This is a negative IgM strip.  Only the 41 band appears. The diagnosis of Lyme should not be based solely on the appearance of the 41 band which itself is considered less specific than several others. 






This is another example of a CDC negative which I consider positive.  The 4 reported bands are all specific. The 39 band -- just 4 percentage points shy of the lab internal cutoff, is perhaps the most specific band, and to the best of my knowledge cross reactivity does not exist. The test is positive by other non-CDC internal laboratory criteria. 












This is a typical LabCorp or Quest report.  As you see, bands are not represented numerically. Bands are reported present or absent.  The results are not determined by a computer which measures pixels, as in the case of MDL, but rather by a technician  eyeballing the strips.  These bands are highly specific, especially the 39 band. I consider this strip likely positive but I would repeat the test from a reference lab before passing judgement. 
























The Blot is highly positive although it is CDC negative.  The CDC requires 5 specific bands not present on this strip.  Several very specific bands are clearly present. 







This report is from IgeneX.  Results are presented is a different format.

IgM: is it chronic Lyme?

I don't care if the bands are IgM or IgG. Lyme IgM bands may persist for years after acute infection.  IgG bands may never appear. 

The 41 band as the sole finding does not lead credence to the diagnosis of Lyme.  It also does not exclude the diagnosis. 


Lyme antibodies do not fit into the traditional mold.  For example, IgM antibodies to the 31 and 34 bands may be present, even though these antigenic proteins do not appear until at least 6 months following initial infection.   The 31 band is associated with outer surface protein A. This protein is expressed with attachment to the tick gut and down-regulates - disappears after infection. It only shows up in the host many months after acute infection.  If you believe that Lyme IgM antibodies disappear within weeks after acute infection, as proposed by the CDC, this paradox is impossible to explain. The cop out that it must be a false positive doesn't fly.

There is a story of repeated by physicians which confuses patients as well as their doctors. This is how the immune system works: IgM and IgG responses are predictable. IgM antibodies appear early, immediately after infection. The better, protective IgG antibodies quickly emerge as  IgM antibodies decline. When the patient recovers from the illness, higher levels of IgG antibodies are present and no IgM is detectable. Therefore in late Lyme, only IgG antibodies are present.

For one thing, "late Lyme" and "chronic Lyme" may not be the same.

Forget this scenario.  Lyme is different.

How do I interpret results?  First, it is important to recognize limitations of these tests. The tests are not automated and require many steps performed by humans, therefore, the chance of error is high. Generally, the results from the 3 labs above are trustworthy. I do not follow any particular guideline approach. Standards which require 2 of the following or 5 etc. lock you in. I would rather interpret results case by case. In my opinion, reading a Western Blot is something like reading an X-ray. A lot of factors are considered --  on the other hand, sometimes Interpreting Blots is like reading tea leaves.  Lab tests are adjunctive and cannot be relied upon as the sole means of diagnosing this complex, enigmatic disorder called Lyme disease. 

Even more important: the CDC standard is for surveillance -- purposes of monitoring the advance of the infection. This should not be relied upon as a diagnostic standard. 










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