Monday, July 18, 2011

13 Lyme Disease Western Blots And Antigen Presentation

I've been thinking of this particular passage on page 504 of the book, Borrelia: Molecular Biology, Host Interaction and Pathogenesis:

"The general picture to have emerged over the years is that IgG and IgM antibodies to the spirochaete develop slowly and are directed against an increasingly diverse array of proteins as infection progresses (Craft et al, 1986; Dressler et al, 1993; Nowalk et al 2006). The earliest responses are to flagellin B (FlaB) and p66, followed by OspC (25kDa) with responses to a number of additional antigens, such as VlsE, fibronectin-binding protein (BBK32), FlaA (37kDa), BmpA (39kDa), and decorin-binding protein A (DbpA) developing as B. burgdorferi disseminates (Coleman and Benach, 1987; Engstron et al, 1995; Bacon et al 2003; Aguero-Rosenfeld et al 2005; Wilske et al, 2007). This temporal pattern is consistent with the notion that the bacterium draws upon an expanding repertoire of differentially expressed proteins once within its vertebrate host, including phased expression of paralogous surface-exposed lipoproteins.
[...]
The clinical ramifications of these observations are significant. Because approximately half of patients with EM do not mount detectable antibody responses to the pathogen, lack of seroreactivity cannot be used to rule out the diagnosis of EM (Steere, 2001; Dananche and Nadelman, 2008). Seroreactivity increases substantially following therapy for EM (Vaz et al, 2001; Dandache and Nadelman, 2008), indicating that killing of the bacterium enhances processing of spirochaetal antigens."

Prior to the 2006 IDSA Lyme disease guidelines, the first set of Lyme disease guidelines which were written contained this specific remark on laboratory criteria for diagnosis:

"Significant change in IgM or IgG antibody response to B. burgdorferi in paired acute and convalescent phase serum samples"

It seems to me that this would still be an important guideline to follow, yet I am aware of a number of stories where patients stated their family doctors gave them an ELISA blood test early after infection, were told it was negative, and because of this, were not given further testing.

This is an inappropriate response, especially given what is known about antibody response in Borrelia burgdorferi infection over time, and given the lack of adequate antibody response early in infection.

Retesting using the western blot several weeks after the initial test - even if negative - makes sense. And if someone is displaying unusually long and moderate-severe flulike symptoms even if there is no EM present, confirmation testing is sensible.

Given this is a serologically progressive infection, why isn't it possible to determine how far long the infection is to some degree (under delayed acute care, at the very least) if the antigens present in a specific order over time? In later infection, it is harder to detect - but doesn't ospA present itself again in neurological infection? Something I have to look into...

13 comments:

  1. CO,

    Based on the information above, it is a bit surprising that there is no way to test for various stages of Lyme disease in humans. I believe such a test is (or will soon be) available for dogs and other animals.

    Doctors who conclude their patients don't have Lyme disease based on one blood test alone may simply not have the necessary training/knowledge to order a repeat test that specifically includes the WB regardless of the ELISA result.

    Other folks may remain seronegative due to inadequate and early treatment (e.g. for a suspected sinus infection) or because of a compromised immune system. The current 2-step ELISA/WB may never produce an accurate result for those reasons alone. If a person mounts some type of immune (antibody) response, s/he will likely have an easier time of getting properly diagnosed -- at least eventually.

    Antibody-based tests do seem to have their limitations, so I'm hoping that other biomarkers might one day be discovered so they can be measured in tests that are readily available to most healthcare providers.

    In the meantime, even improving on antibody-based testing would be a welcome development.

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  2. Rita,

    Was wondering if you saw this entry I posted a while ago:
    http://campother.blogspot.com/2011/06/paper-fluorescent-bead-based-multiplex.html

    There is a test available for dogs, and supposedly a test that is already available for people in Europe. I don't see that they're that much better than what's already on the market in a number of ways, though, and I want to know more about them.

    It seems to me that based on this and other research I've done that repeat testing in order to track IgM and IgG antigens is really a good idea and gives clinicians an idea of what's going on with an existing infection and may provide evidence of a persisting one. I've been wanting more information on the IDSA's claim that continuing to have positive tests does not mean that one currently has an infection - what evidence do they have to substantiate this as a general rule?

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  3. CO,

    (lol) Your blog entry is likely where I got the idea that tests for animals are better than what we currently have available for humans in North America.

    Yes, I do agree that repeat testing has value and needs to be carried out on a routine basis. Let the scientists/researchers figure out all the details, but in the meantime, let's gather some real data (in the form of blood tests) instead of continuing to speculate about so very many things.

    There seems to be little evidence to support much of what is widely accepted as fact in mainstream medicine. Evidence-based or even science-based medicine is great, but the evidence and science seem to be missing when it come to quite a few medical practices.

    The more I learn about tick-borne illnesses, the more questions I have. So many that I can no longer keep track of them.

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  4. This section is part of what I copied and had considered taking to my regular MD, the one who told me I had a false positive.

    (My test through Quest: IgM 41 & 23, IgG 66)

    I have read stories about people who seroconvert to a full blown western blot many months later..and then they are considered a case per CDC criteria.

    I don't know if you remember the chat between Dr. Zemel and Dr. Cameron...

    Dr. Zemel: "Dr. Cameron is correct: early treatment with antibiotics may blunt antibody response, but at that point, no further treatment is needed."

    So, even with a change in paired sera, I don't think it would matter to people like Dr. Zemel. He would say, oh you took your XX days of antibiotic. You don't have lyme anymore.

    The problem is, we can't force people to think for themselves and read for themselves, but, just look around, there are many hot topics with the same effect on people.

    It's an ingrained dogma that they refuse to look beyond, even when the data is readily available.

    I still think what could change this is more research on the L-forms, and identifying them in humans.

    http://www.personalconsult.com/articles/LymeDiseaseCystsLFormsBlebs-2.pdf

    under #10 I was heartened to see this: "When transferred to BSK II medium, the cystic forms reconverted to spirochetes in relation to their age and type of induction treatment."

    That made me wonder, if you kept them in L-form long enough would they lose the ability to revert, and the ability to replicate?

    ReplyDelete
  5. jjbluemountain,

    You wrote, "So, even with a change in paired sera, I don't think it would matter to people like Dr. Zemel. He would say, oh you took your XX days of antibiotic. You don't have lyme anymore."

    Well, that's part of the issue for me as well. I don't think you can definitely say that infection is over - particularly in early infection, and with a prophylactic amount of antibiotics like the two dose doxycycline. I'm on Dr. David Volkman's side on this. Also, if one has had a highly disseminated infection that has spread to the CNS, it's possible that one's tests won't reflect that infection and to get the most accurate 'read' from the CSF, one has to test a patient in the first 2 weeks after the bite.
    At that point, there may or may not be an EM.

    There's evidence that staging (Stage I, Stage II, Stage III) at each of its cut-off points is pretty arbitrary; dissemination to the CNS can happen within the first week after the tick bite. So what happens if someone has a CNS infection but the antibody test is a negative ELISA early on? A good doctor would retest with the western blot when the patient continues to have symptoms and treat them for neuroborreliosis. One that doesn't know how to assess the situation would tell the patient "you can't have Lyme; the ELISA was negative". I think the doctor needs to be more of a detective and also see how the patient responds to early treatment. I think an early relapse is easier to recognize and treat than a later one - that's when the controversy kicks in.

    You said, "I still think what could change this is more research on the L-forms, and identifying them in humans. under #10 I was heartened to see this: "When transferred to BSK II medium, the cystic forms reconverted to spirochetes in relation to their age and type of induction treatment."

    (As an aside, in the future I request that you post direct references to papers rather than post it from doctors or the IDSA's site - unless you are specifically looking to quote that doctor or the IDSA. Just trying to ask that everyone looks for the most general and ongoing accessible official source for documentation where possible, because as time goes on, individual doctors' websites and those of the IDSA are more subject to edited pages and dead links than PubMed. Thanks.)

    Regarding the above, between reading the Borreliosis textbook and reading these publications, have you come across in vivo studies as to how the organism behaves then? One of the difficulties in knowing how to address the different forms of spirochetes is that different media produce different results and behavior in vivo can be different. It's even different for different host systems. And behavior in vivo where spirochetes are confronted by both the immune system and antibiotics are yet two additional factors not found in vitro.

    So I'm not certain how much emphasis to place on these forms. Dr. Stephen Barthold, who has studied Lyme disease in murine models, hasn't found the issue of form to be relevant to whether or not the spirochetes found after antibiotic treatment are viable and capable of reproducing. I don't know how relevant it is either, even though the Lyme patient community has often cited form as relevant to their being treatment resistant.

    What has to be established is their ability to continue to be infectious and if they cause symptoms, what the mechanism for that is.

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  6. "Regarding the above, between reading the Borreliosis textbook and reading these publications, have you come across in vivo studies as to how the organism behaves then? "

    I have not specifically looked yet. I think I saw some information out there regarding primates...I need to check further.

    I would like to see if anyone has labeled parts of borrelia and tried to find them in people.

    The video of the fluorescent spirochetes in the mice ears was fascinating.

    "It's even different for different host systems. And behavior in vivo where spirochetes are confronted by both the immune system and antibiotics are yet two additional factors not found in vitro."

    very true, I just think we can't discount anything at this point. If it behaves a certain way in vitro, then we keep an open mind to the possibility that it could do the same thing in vivo, although maybe not!

    "So I'm not certain how much emphasis to place on these forms. Dr. Stephen Barthold, who has studied Lyme disease in murine models, hasn't found the issue of form to be relevant to whether or not the spirochetes found after antibiotic treatment are viable and capable of reproducing."

    I'm not certain either. I haven't seen enough information to convince me either way.

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  7. http://www.ncbi.nlm.nih.gov/pubmed?term=wormser%20epitope%20mapping

    Is this what you mean by posting the link to an article/paper? Did I do it right this time? :)

    This is what I mean about finding the borrelia parts.

    I don't have access to the whole paper yet, but I am wondering if they can identify WHERE the epitopes are located that are causing this immune response (this paper looks to only be about mapping the epitope on the bacteria).

    Also, I always love how they say "despite antibiotic treatment". It reminds me of the IDSA doc I went to when I was still convinced I had a bad sinus infection. He said I was getting better with time, even though I told him I was getting better with antibiotics.

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  8. jjbluemountain,

    Yes, that's better. In the future you can also make any link an active link by using the "a href" tag markup mentioned just above the post comment box. That way, anyone seeing your link can click on it rather than copy & paste your link into a browser.

    Re epitope locations: How much detail do you need? It's good to know which are activated and when in specific hosts - the differences are important.

    You probably want to check out earlier papers including refs in the one you posted. See:

    Dominant Epitopes of the C6 Diagnostic Peptide of Borrelia burgdorferi Are Largely Inaccessible to Antibody on the Parent VlsE Molecule
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2044495/
    and
    Epitope Mapping of the Immunodominant Invariable Region of
    Borrelia burgdorferi VlsE in Three Host Species
    http://iai.asm.org/cgi/reprint/68/4/2349.pdf

    Let me know if these answer your question or if you're looking for something else.

    Re "despite antibiotic treatment": It is an oft-used phrase and I wish that there were more qualifiers on it when it's used. What does the phrase mean relative to the rest of the paragraph or author's context? Does it mean antibiotic treatment of the specific class used would be expected to handle it and didn't - or is the author thinking that any antibiotic would not work because the mechanism producing symptoms is not infectious?

    And here's the thing with this, overall: Researchers have their own shorthand for what they mean when they write such phrases within their own papers so that other researchers do not even have to ask what a phrase means. Those without the same knowledge base are more likely to see ambiguity in certain phrases being used. This is why it's a good idea for researchers to hand their papers to others outside their field for proofreading because by not understanding, they may ask questions which encourage more clarity in their writing.

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  9. Everyone interested in this topic may want to check out this oldie-but-goodie paper:

    Antigens of Borrella burgdorferi Recognized during Lyme Disease
    Appearance of a New Immunoglobulin M Response and Expansion of the
    Immunoglobulin G Response Late in the Illness
    by Joseph E. Craft, Duncan K. Fischer, Grant T. Shimamoto, and Allen C. Steere
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC423723/pdf/jcinvest00109-0086.pdf

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  10. This comment has been removed by a blog administrator.

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  11. Welcome Irina,

    I think your English is okay. If there is another language you speak you are more comfortable using - such as German, Dutch, or Spanish - please let me know. It will take longer on this end to translate but can be done.

    Did a doctor order the blood tests for Borrelia? If a doctor knows the test results are positive for Borrelia then that doctor should be giving high doses of antibiotics to your relative.

    If it is a new infection, usually 300-400 mg of doxycycline daily for a month to start. If your relative has been sick with Borreliosis for months or years, then IV antibiotics may be needed - usually daily ceftriaxone.

    I am not a doctor, I am a researcher, so I cannot give medical advice. I can tell you what doctors do in general - and that different guidelines for antibiotics to use for treatment can be found online.

    If you are worried that your relative's Borreliosis disease will turn into Alzheimer's disease in the future, I don't think that has to be the case. People get Borreliosis, get treated, and get better.

    I know Dr. Miklossy from Switzerland has been doing research on the potential relationship between spirochetes and Alzheimer's Disease, and she is looking at all kinds of spirochetes including ones which live in one's mouth. Other researchers have been looking at other causes for the disease - such as environmental and genetic factors. But it may be there is more than one factor which gives rise to Alzheimer's Disease - even in an infection model.

    The best thing for you to do now is make sure your relative gets treated properly as soon as possible for Borreliosis and also gets examined, tested, and treated for any other tick-related infection such as Babesiosis, Ehrlichiosis, and others.

    Hope this helps. If there is anything I wrote which you do not understand, please ask and use another language if you need.

    Best to you, and thank you for reading Camp Other.

    Camp Other blog

    ReplyDelete
  12. Good afternoon,

    Thank you very much for all.
    The Alzheimer disease was detected before B. and I don't know when she contacted Borrellia (years, perhaps). We do not know the Alzheimer's origin.

    I read on Internet that many spirochetes gives Alzheimer symptoms but I do not understand the followings:

    1. the spirochetes grows the content of amiloyds in the brain ?
    or
    2. the spirochetes grows the content of other components in the brain ?

    If 1 is true, I understand that we may do anything for loss of amiloyds existing here, but, the treatment antiB. stops the growing of concentration of amiloyds, if B. is the only one cause ?

    If 2 is true, by treatment, the loss of these components is possible or the stopping, only?

    About other analysis for Babesiosis or.. a fiew chances, here.


    Best regards,
    Irina

    I kindly ask you to erase my yahoo's addressee from the blog, but we remain in contact on the blog. It was for your replay by e-mail.

    ReplyDelete
  13. Irina,

    I had to delete your original comment because I cannot edit others' comments. Apologies.

    Thank you for explaining in more detail what is happening with your relative.

    Scientists are still trying to figure out what causes Alzheimer's disease (AD) - it may be a combination of causes that produce AD.

    I will try to answer your questions based on my understanding of what you wrote.

    Based on research to date:

    1) Spirochetes themselves do not grow amyloid plaques in the brain.

    2) It is speculated that something triggers a response that leads to amyloid deposits. What this trigger is depends on who you talk to - some say amyloid deposits have a genetic basis and build up before one develops AD. Some say that the binding of part of a protein (related to amyloid-beta) to a certain receptor on neurons causes a degenerative process. There are other hypotheses that have been considered, too, such as tau hypothesis (to greatly simplify it, the tau protein in the brain breaks down and causes damage) and also the idea that myelin breaking down causes AD.

    There is a hypothesis that infections of different kinds may trigger AD. This is what Dr. Miklossy thinks happens based on her studies and a few other studies. She is not the only one who has this kind of hypothesis - other researchers have looked at a virus like HSV-1 as being a possible factor in AD. Also, researchers have looked at C. pneumoniae (see http://www.biomedcentral.com/1471-2202/11/121) as a possible factor in AD.

    Most of the studies Miklossy discusses focus on oral spirochetes (Treponema spirochetes) and not Borrelia spirochetes. Why Treponema spirochetes are found in the brains of some people and not other people is not clear - everyone has these Treponema spirochetes in their mouths.

    So far, the best drugs available for slowing the progression of AD are drugs like Aricept and Nameda, each of which operate on a different mechanism. Only a doctor can best decide which medicine should be used in an AD patient.

    If your relative already has a definite diagnosis of both Alzheimer's disease and an untreated case of Borrelia, a doctor needs to decide which medication to dispense to treat both conditions. Self-treatment is not advised and a bad idea - these are complex conditions - plus you need to avoid serious drug reactions.

    Hope this helps.

    Camp Other blog

    ReplyDelete

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