0 Reader Mail Bag: What Lyme Disease Research Is Needed?

A reader, Misty, recently commented on my request for topics for discussion this week:

"I like all your ideas for topics - and hope you'll be able to continue posting. I love your "blog - it is one of the most sane Lyme sites on the web, if not the most sane and balanced.

What I wonder is - do we have enough information and diagnostic tools to be able to design useful studies on Lyme?

- we can't tell reliably who has it or doesn't
- the manifestations of Lyme in each person can be different and based on complications of co-infections and the genetic predisposition of the person to exaggerated inflammatory response
- then there is the pesky post-Lyme-Syndrome/Chronic Lyme issue of whether there is infection or post-infection inflammation

So, you may have covered it already, but I am interested in hearing about ideas for scientific studies - where is the research most needed?"

thanks,
misty

Well, Misty, addressing your questions and points:

I think we can design useful studies on Lyme disease even without being capable of accurately testing every patient who has Lyme disease. Improving serological testing and being able to accurately assess whether one has or does not have Lyme disease at present are only two pieces of the bigger picture, and there are more angles from which to approach the Lyme disease problem.

Research that can be useful in gaining a better understanding of what Borrelia burgdorferi and other Borrelia do is important to understanding how to effectively diagnose and treat infection and perhaps distinguish between patients who are affected by Lyme disease and those who are affected by a different condition.

Here's a few ideas I have on what to consider for further study:

1) Do a comparative study which looks at the proteins in the CSF of patients with chronic Lyme disease versus patients with late stage untreated and patients with acute Lyme disease.

Earlier this year, we've seen the study where hundreds of proteins were found in the CSF of patients with post-treatment Lyme disease symptoms and compared against patients with Chronic Fatigue Syndrome. The protein profile for each group was different, and each group's profile differed from healthy controls.

Let's take this study one step further, and see if there is a protein profile that distinguishes between patients who were designated as suffering from post-treatment Lyme disease symptoms and those who are  late stage and newly infected.

The outcome of this study may shed some light on what markers are present for different stages of the disease. Having different markers for different stages of the disease may help guide better test research and development.

References:
Steven E. Schutzer, Thomas E. Angel, Tao Liu, Athena A. Schepmoes, Therese R. Clauss, Joshua N. Adkins, David G. Camp II, Bart K. Holland, Jonas Bergquist, Patricia K. Coyle, Richard D. Smith, Brian A. Fallon, Benjamin H. Natelson. Distinct Cerebrospinal Fluid Proteomes Differentiate Post-Treatment Lyme Disease from Chronic Fatigue Syndrome. PLoS ONE 6(2): e17287. doi:10.1371/journal.pone.0017287 http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0017287

2) Conduct longer term in vivo GFP and/or iRFP studies on mice and other mammals.

In this GFP protein study on mice, spirochetes' motion was monitored in vivo rather than in vitro (go to link to watch video of spirochetes attaching to endothelial walls). Rather than study it for as short a period of time as was done, a longer time frame for study as well as multiple studies over time in the same hosts would be educational.

With longer term imaging, one can see if spirochetes become intracellular and for how long. One can see which parts of the body they travel to and see them hide in immunological niches. One can see how likely different strains are to enter the CNS and how quickly they enter the CNS post-inoculation. (We already know specific strains are more neurotropic than others, but how serious a problem is this for the host? Does it depend on the host animal?)

Perhaps a GFP or iRFP study on mice could also be combined with an antibiotic treatment study. If we can trace the activity and polymorphic state of spirochetes in vivo, then we can see if antibiotics of specific types can affect "cyst"-like forms of spirochetes in vivo, too.

3) Repeat Klempner intracellular studies with a longer observation time and longer ceftriaxone infusion.

Also - give two weeks' ceftriaxone then provide no treatment for a few months. Try a different duration of ceftriaxone. Recheck the host animal for signs of infection at 3 months, 6 months, a year, then two years.

How can an in vivo study of this issue be completed?

References:
Kostis Georgilis, Monica Peacocke, and Mark S. Klempner. Fibroblasts Protect the Lyme Disease Spirochete, Borrelia burgdorferi, from Ceftriaxone In Vitro. Journal of Infectious Diseases. Vol. 166, pp. 440-444. 1992.
Mark S. Klempner, Richard Noring and Rick A. Rogers. Invasion of Human Skin Fibroblasts by the Lyme Disease Spirochete, Borrelia burgdorferi. The Journal of Infectious Diseases. Vol. 167, No. 5 pp. 1074-1081. May 1993. http://www.jstor.org/pss/30112679

4) Use new maltodextrin enhanced imaging study in animal subjects (and later people) to see where bacteria is.

This is a very new imaging method, but the advantages are clear: Maltodextrin is viewed by pathogenic bacteria as food, whereas regular mammalian cells (human, mouse, other) and even commensal or friendly bacteria in the gut do not view maltodextrin as food and they work to eliminate it.

With the addition of a maltodextrin contrast agent, one should be able to see where pathogenic bacteria are present in the body in vivo and do so safely.

And there's more:

"In experiments using a rat model, the researchers found that the contrast agent accumulated in bacteria-infected tissues, but was efficiently cleared from uninfected tissues. They saw a 42-fold increase in fluorescence intensity between bacterial infected and uninfected tissues. However, the contrast agent did not accumulate in the healthy bacterial microflora located in the intestines. Because systemically administered glucose molecules cannot access the interior of the intestines, the bacteria located there never came into contact with the probe. 

They also found that the probes could detect as few as one million viable bacteria cells. Current contrast agents for imaging bacteria require at least 100 million bacteria, according to the researchers. 

In another experiment, the researchers found that the maltodextrin-based probes could distinguish between bacterial infections and inflammation with high specificity. Tissues infected with E. coli bacteria exhibited a 17-fold increase in fluorescence intensity when compared with inflamed tissues that were not infected."

All of these items in bold are of particular interest to those wishing to see where Borrelia burgdorferi is present during infection. If - as a number of researchers have stated - Borrelia burgdorferi are actually low in number and produce high amounts of inflammation in tissues, maltodextrin contrast should be able to confirm this finding. We'd also have a better idea of where the bacteria is in vivo without having to do a tissue biopsy, and be able to detect biofilms if any have formed.

Initial studies should be conducted on animal models, and if proven safe and effective, I see no reason why human studies wouldn't follow.

References:
Xinghai Ning, Seungjun Lee, Zhirui Wang, Dongin Kim, Bryan Stubblefield, Eric Gilbert, Niren Murthy.Maltodextrin-based imaging probes detect bacteria in vivo with high sensitivity and specificity. Nature Materials, 2011; DOI: 10.1038/nmat3074
Scientific American: http://blogs.scientificamerican.com/lab-rat/2011/07/25/making-bacteria-visible/
Science Daily: http://www.sciencedaily.com/releases/2011/07/110718121605.htm
Nature: http://www.nature.com/nmat/journal/v10/n8/full/nmat3074.html

5) Complete more treatment studies on patients with documented late stage Lyme disease and coinfections such as Ehrlichiosis and Babesiosis.

If it's problematic to differentiate between those who suffer from a chronic, persisting infection and those who suffer from an autoimmune disorder, then circumvent the issue by finding people who are truly late stage, untreated Lyme disease patients who have coinfections and study how long it takes for them to get well on combination treatments.

Many Lyme patient activists promote more studies for those of us suffering from persistent post-treatment symptoms when perhaps it is more advantageous to first push for the study of patients who have never been treated and have evidence of late stage symptoms. There are far more studies on acutely infected patients than there are on late stage patients, and this needs to be addressed, I think, in order to bridge the gap between acute cases and post-treatment cases (chronic Lyme; PLDS) and work past any controversy.

6) Run comparative studies on all labs which conduct Lyme disease tests - C6/ELISA and Western Blot IgM and IgG.

Test all existing labs for sensitivity and specificity for various strains including Borrelia lonestari and miyamotoi - include the relapsing fever Borrelias. It would be informative to know how all the labs perform and why they receive the results they do.

These are just some of the ideas I have on studies which could be conducted that give us more answers.

Regardless of these suggestions, one has to be aware of the limitations of using animal studies to model what happens in human infection. For one thing,  even non-human primate studies may not show evidence of a Borrelia burgdorferi brain infection, even with an N40 strain of Bb which is neurotropic. For another, mice do not get brain infections and are thus a poor model for studying neuroborreliosis.

References:
Ramesh, G., Borda, J., Dufour, J., Kaushal, D., Ramamoorthy, R., Lackner, A., & Philipp, M. (2008). Interaction of the Lyme Disease Spirochete Borrelia burgdorferi with Brain Parenchyma Elicits Inflammatory Mediators from Glial Cells as Well as Glial and Neuronal Apoptosis. American Journal Of Pathology, 173 (5), 1415-1427 DOI: 10.2353/ajpath.2008.080483
Diego Cadavid, Tim O'Neill, Henry Schaefer, and Andrew R. Pachner (2000). Localization of Borrelia burgdorferi in the Nervous System and Other Organs in a Nonhuman Primate Model of Lyme Disease.Laboratory Investigation, 80 (7), 1043-1054

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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...

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4 The FDA Vault: Dr. Vijay Sikand On Lyme Disease & Lymerix

You know how I've said you can never guess what you're going to come across when you surf the net looking for other things?

Well, I came across this FDA transcript for a discussion on the Lymerix vaccine back in 1998. The original transcript contains an endorsement for the vaccine from the founder of Lyme Disease Foundation (LDF, not to be confused with the ADLF), who later determined the vaccine needed to be pulled from the shelves based on adverse reaction reports.

I'm setting aside any discussion of the Lymerix vaccine for now and just posting this statement from Dr. Vijay Sikand, an MD who practiced in Lyme, Connecticut... (items of interest are in bold)

UNITED STATES OF AMERICA 

DEPARTMENT OF HEALTH AND HUMAN SERVICES
FOOD AND DRUG ADMINISTRATION
CENTER FOR BIOLOGICS EVALUATION AND RESEARCH
VACCINES AND RELATED BIOLOGICAL PRODUCTS ADVISORY COMMITTEE MEETING
Tuesday, May 26, 1998 

  
The meeting took place in Versailles Rooms I and II, Holiday Inn, 8120 Wisconsin Avenue, Bethesda, Maryland at 9:00 a.m., Patricia L. Ferrieri, M.D., Chair, presiding.  


VIJAY SIKAND, M.D., Sponsor Rep
:

"...As I was just saying, I included research in Lyme disease as part of a primary care practice for a number of years. In early 1995, 1,200 volunteers came to my office to enroll in the SmithKline Beecham vaccine trial which we are discussing today. Almost three and a half years later now, greater than 92 percent of those patients are still providing me with clinical follow-up.
        
Why do we need a vaccine for Lyme disease? It has been almost a quarter century since Lyme disease was first described as an emerging infection in this country. During these years a number of factors, epidemiologic factors and clinical factors, have resulted in considerable morbidity in burgeoning numbers of patients.This burgeoning load of disease as well as the increasing number of patients thus set the stage for prevention of this disease with a vaccine.Today, I will present to you some of the factors in a brief synopsis illustrating the need for a vaccine for Lyme disease.The illustrations which I will present to you, some of them are from my private practice and some of them are from the vaccine study.

The first factor is an epidemiologic factor,and this has already been discussed by Dr. Schoen. And that is that there is indeed a progressive increase in incidence of Lyme disease. The second factor also epidemiologic is the relentless geographic spread of this disease. There are new endemic areas being created annually and the disease burden is indeed growing.
        
The ineffectiveness of preventive measures which we attempt to practice is another important factor. We have tried various chemical and other means. Why have preventive measures, which are indeed important, not been effective in preventing an increase in cases of Lyme disease?  And before I answer that question, let me underline the fact that I indeed believe it is important that we continue to practice preventive measures because of co-infection with other illnesses besides Lyme disease. 

One obvious reason is that it is very impractical to practice certain protective measures.  This individual in the Lyme, Connecticut area desires to do some outdoor work and does not want to be bitten by a tick. But the point is it is very difficult to ask children or anybody else for that matter to tuck pants into socks, et cetera, in the middle of July and August when the ticks are questing. We can certainly check our pets, but checking one's dog is indeed a Sisyphean task when the dog goes in and out of the house all day long. Probably the best protective measure, I think, in preventing Lyme disease is checking for ticks.  Unfortunately, kids will only allow you to do this up to a certain age. And of course one must be vigilant with oneself.

More specifically, I think one of the important reasons to consider when thinking about why protective measures are difficult to utilize and be effective in preventing this disease is simply the nature of the Ixodid tick bite itself. The bite of this tick when it is infected transmits not only saliva infected with Borrelia burgdorferi, but the saliva also contains certain anti-inflammatory substances which have an anesthetic effect.The end result of that is that tick bites in general are not noticed. In one study, over 80 percent of the patients who presented with definite Lyme disease did not remember a tick bite. It is therefore very hard to correlate the incidence of definite Lyme disease cases with preceding tick bites, and this is well known.

Furthermore, as has been eluded to earlier, the recurrence of disease in individuals is also well known. Unfortunately, in the majority of patients, the vast majority of patients, natural infection with Borrelia burgdorferi does not confer protective immunity. 

Difficulties in clinical diagnosis of this disease are also well known, and it is not my place today to give you an overview or detailed presentation of the clinical aspects of Lyme disease.However, a couple of issues that do spring up and which I would like to address are as follows. In particular, the specter of asymptomatic infection is something that troubles me a great deal and troubles a great number of my colleagues who need to treat Lyme disease. The obvious analogy with syphilis infection with Treponema pallidus is there to consider. It is well known that Borrelia burgdorferi indeed after asymptomatic infection can lurk or secrete itself in certain areas of the body, perhaps the central nervous system or perhaps the joint spaces, only to reappear months or maybe years later in the form of late stages of illness which are harder to diagnosis and treat.

In terms of the variability of Lyme disease, it is indeed a very variable infection, if not a very complex infection.  In its very simplest form, it is erythema migrans, well localized, which we can all recognize and which we can all easily treat and from which most patients can get better. However, erythema migrans is not a single beast. Certainly this is the one which we easily recognize and which I just referred to.Before I continue with further slides, let me point out that the erythema migrans lesions you are about to see are all biopsy lesions which were laboratory proven to be caused by Borrelia burgdorferi.

Sometimes erythema migrans can present as a pustular lesion as is this one in the popliteal fossa inviting the scalpel of a surgeon. Sometimes the lesions are vesicular in nature, inviting a diagnosis perhaps of herpes simplex infection.  Sometimes our round lesion is actually triangular. Sometimes it doesn't even look round or red at all and invites a diagnosis of an intertriginous fungal infection in the groin of this patient who was biopsied and proven to have Lyme disease. Sometimes the lesion is more plaque-like, inviting diagnosis of nummular eczema, psoriasis, or other similar lesions. Sometimes it is in unusual locations. Sometimes it is large like this one. Sometimes it is small with satellite areas. Sometimes it is multiple, appearing almost like urticaria or erythema multiform. Sometimes, as in this individual who was a placebo recipient in the Lyme 008 SmithKline Beecham trial, it presents with other manifestations of early dissemination. This individual came in mainly because he was concerned about his face and it felt kind of funny and it was weak on one side. When I asked him whether he had had any unusual rashes, he said oh do you mean this one, and he showed me his arm with that EM. This is simply to illustrate the infranuclar 7th nerve palsy with which he presented. This patient, by the way, had no history of a tick bite or any unusual antecedent illness which he could remember.

The next slide is the electrocardiographic tracing of a 37-year-old mom from Lyme, Connecticut, mother of three. Generally healthy and no medical problems. Early on the day that this electrocardiogram was taken, she went to her local health club and did her usual work-out, which went fine. However, when she came home that day, she noticed that she had some palpitations, a little shortness of breath, malaise, and things just didn't seem quite right, but she wasn't sure what. When her husband came home, she told him that maybe she had worked out a little bit too hard at the club. A few minutes later, he was reading the newspaper in an armchair and he heard a thump on the floor above. He ran up the stairs to find his wife unconscious briefly on the floor and called 911. On arrival at the emergency department, the patient presented with this tracing, which in retrospect was a superventricular tachycardia representing an escape rhythm. 

There was fortunately a very vigilant emergency physician who didn't understand quite why a 37-year-old healthy woman had completely passed out, and she had what was a relatively benign rhythm at that point. But he was wise and admitted her to the coronary care unit for further monitoring. Late that night and the early hours of the following morning, the CCU nurse noted that the patient had gone through progressive degrees of AV block culminating in complete atrial ventricular dissociation. A cardiologist was summoned. He inserted a temporary transvenous pacemaker. The patient was started on intravenous antibiotics for about a week in the hospital followed by a few more weeks as an outpatient.This patient also had no history of a tick bite.

Besides the difficulties in clinical diagnosis, we are all aware that quandaries in laboratory diagnosis are rife.We rely pretty much on serologic testing in the United States today to assist us in diagnosing Lyme disease. Unfortunately, serologic testing, as with other infectious diseases, provides only indirect evidence of infection. When we order a serologic test, it just tells us that the patient has been exposed to Borrelia burgdorferi and doesn't tell us whether the infection is active or whether it is a past infection. It is probably worth noting, since I have learned a lot, that we don't have the clinical luxury in private practice that we had in the SmithKline Beecham trial in which we had baseline sera on all the patients who enrolled so that when they presented with symptoms, we could draw acute and convalescent serologies so as to compare them with each other and with baseline to better understand what symptoms they are presenting with. But your average physician in the office just can't do this. A patient comes in with symptoms or signs of Lyme disease and you have to make a clinical diagnosis and it is not always easy and serology doesn't help. The fact that in particular the ELISA creates a great deal of false positive results is also problematic. 

In particular and commonly in infectious mononucleosis and other spirochetal disorders, even healthy people, juvenile rheumatoid arthritis and other autoimmune disease all can produce false positive results. Indeed, even with Western blotting recent reports have shown that infection with the agent of human granulocytic Ehrlichiosis can cause false positive Western immuno-blots.The false negatives that we deal with are generally caused by use of serology testing in patients who have early Lyme disease and in whom the serologic response with immunoglobulin M has not occurred to the extent to which it can be measured.

What do we have in the way of direct testing to try to see if the organism itself is actually there or evidence of it? Well, culture and PCR are what are out there right now.  However, these are unreliable and impractical. Culture and PCR are certainly not warranted for the diagnosis of erythema migrans. The polymerase chain reaction is indeed sensitive in joint fluid. However, the diagnosis of Lyme arthritis does not require PCR testing since serology is almost invariably positive at that stage. Clinical conditions such as complex neurological conditions when a test like sensitive PCR would be useful, unfortunately cannot be diagnosed that way because PCR and indeed culture are not sensitive for cerebrospinal fluid, nor are they sensitive for urine, blood, and other body tissues when later in the disease one might care to employ these techniques.

Finally, there are indeed many dilemmas in therapy. In particular, untreated or inadequately treated Lyme disease may lead to the chronic morbidity with which we are very familiar. Most commonly arthritis and the not common but complex neurological syndromes are what often result and which confront the primary care physician in the office diagnostically and therapeutically.These particular outcomes result in much more intensive, long-term expensive therapy, often in the form of long-term intravenous antibiotics. These are the patients who often are refractory to treatment. Indeed, these are the patients in whom symptoms seem to persist despite what we have given in terms of adequate antibiotic therapy by any known measure.

            In conclusion, we need a vaccine for Lyme disease because it is increasing in incidence and geographic spread.We need a vaccine for Lyme disease because there are problems in clinical diagnosis, its laboratory evaluation, and its treatment. We need a vaccine for Lyme disease because preventive measures are unfortunately ineffective. Lyme disease is indeed vaccine preventable. Availability of this vaccine would lead to a significant reduction in chronic sequelae and substantive morbidity.  Lyme vaccine is thus a critical new public health approach to the primary prevention of Lyme disease in the United States. Thank you very much."

Dr. Sikand and Dr. Steere were at the same meeting. It's obvious Sikand knew Lyme disease could lead to serious late stage Lyme disease and chronic persistent symptoms of infection. If it could be easily resolved with 2-3 weeks of antibiotics, would they have spent this much time, effort, and money on developing a vaccine and running clinical trials?

Everyone at this meeting - including Lyme disease patient advocates - wanted a vaccine. That it would lead to adverse events in some patients and be pulled from the market later was not what everyone thought at first - at first it was welcomed as a solution to preventing the worst of what Lyme disease could do to a person because it was hard to diagnose and treat.

1998. Thirteen years ago.

How much have things changed since then?

Why are we still looking at the same problems and issues?

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0 IOM Summary Report: Neuroborreliosis Notes #2

This is Part 2 of part 1, IOM Summary Report: Neuroborreliosis Notes...
(Read that first...)

After Dr. Stephen Barthold's presentation, the following discussion came up:

"Another participant questioned how neurologic symptoms occur if the bacterium is just in collagen,even if it is associated with neural tissue. Barthold noted that mice do not get central nervous system disease, possibly because they don’t have much connective tissue in their brain. However, central nervous system disease is seen in larger mammalian species that have more collagen in their meninges and perivascular spaces. Under those circumstances, Barthold has observed spirochetes in the collagenous areas and along the perivascular spaces into the brain. Because there is a dearth of good analysis of human neuroborreliosis cases, it is not known if the spirochetes are located in other areas.  He noted that a tissue bank or biorepository would be very valuable to allow for these types of analysis."

Has Dr. Barthold talked to Dr. Alan MacDonald at all? I have to wonder... aren't there other tissue banks out there which already have neuroborreliosis samples?

So all these people - a portion of which knew they were bitten by ticks - have cognitive and neurological symptoms, but there is little good analysis of human neuroborreliosis cases.

Why is that?

Does it have anything to do with how difficult it is to determine whether or not a patient has neuroborreliosis, especially once you're read the European data on testing the CSF for IgG antibodies and realize that the initial positive rate is at it's best within two weeks of clinical presentation (assuming this means symptom onset)?

When they say there isn't enough data on human neuroborreliosis... let's start with this chart:

No news is good news?

I note that there also is no independent measurement for Late disease-Neuroborreliosis  in this table, either. Just arthritis and the measure for culture is labeled "anecdotal". So data is lacking there, too. (I have to wonder why the author used "ancedotal".)

But then there is always two-tier testing, which is indirect detection testing (does not test for the organism itself, but tests for antibodies to it):

As you can see from this chart, depending on which kind of testing you use (ELISA, two-tier Western Blot, or this other proposed test), supposedly serological testing for early Lyme disease is poor, and early disseminated neurological infection is better. Not perfect, but better.

Is that late neurological and arthritis disease detection rate correct? I want to see the cited research (this requires finding the original full text publication) before commenting more on it. But I will say that even with the correct distribution of findings here,  neuroborreliosis that occurs within the first week after being bitten is not going to show up in the early group, and 13-37% of early disseminated neuroborreliosis cases are missed, depending on the test set.

Note on page 7-4 that:

"The C6 testing protocol has performed comparably in accurately detecting the presence of antibodies to B. burgdorferi in sera of patients with acute EM, but was slightly less effective in the case of neurological Lyme disease."

And also, this is an important point, related to the first chart at the top and information in my previous neuroborreliosis notes post:

"One scientific gap is the testing of cerebrospinal fluid for antibodies. Europeans measure intrathecal production of antibodies by measuring antibodies in CSF and comparing these results against the concentration of antibodies in the serum to produce a ratio. U.S. Scientists have not had a sufficiently large population in which to evaluate the efficacy of this approach because fewer cases of neuroborreliosis are documented in the United States as compared to Europe and CSF sampling is not routinely done in patients with Lyme disease. The absence of this type of testing is a gap in diagnostics for neuroborreliosis caused by B. burgdorferi in the United States."

Is this the case - that the gap in diagnostics for neuroborreliosis is to be blamed on the bacteria? I think that's unfair to the bacteria. As far as I can see, what's happened is that the fact of neuroborreliosis is not something that has been discussed or emphasized when pathogenic Borrelia are neurotropic to varying degrees. Neurological symptoms should be included when educating doctors, nurses, and patients about the disease, and not sidelined to "that only happens in European strains". It doesn't, and besides, Americans do sometimes visit Europe. And hey, if B. garinii and B. afzelii are beginning to show up in southern ticks, well, maybe in a while you'd be seeing more cases of early neuroborreliosis anyway.

I don't know about you, but many Lyme disease patients I've spoken with only got an ELISA test for Lyme disease from their primary care physician, and when that came back negative, there was no further testing.

CSF testing when I had early neurological symptoms is not something I was offered, either.

Which is ludicrous when early serological testing is especially prone to not detecting an infection. What should happen is if the person has a lot of neurological symptoms shortly after a tick bite, they should have a LP. And also the Western Blot. Repeated Western Blot testing which shows an increasing and changing serological profile would be useful in demonstrating antibody response.

It is also stated elsewhere in the summary report:

"An increment in immunoreactive bands is observed in the IgG immunoblots of sera of patients with neuroborreliosis and Lyme disease arthritis."

Saying that Borrelia burgdorferi doesn't cause neuroborreliosis isn't true. Saying it's rare isn't a helpful statement, and aside from utility, it's not even clear how rare it is because we simply lack that data. We don't know. What we have is a guess.

Referring to some studies (there are more studies on neuroborreliosis in the paper):

Page 7-15

"Turning to the literature pertaining to patients with chronic persistent symptoms, Fallon noted a number of areas need additional research. A European study compared patients with neurologic Lyme disease to those with erythema migrans 3 years later and found that 50 percent of those with neuroborreliosis experienced persistent symptoms versus 16 percent of the EM patients (Vrethem et al., 2002). These results suggest that follow up studies on chronic symptoms, rather than focusing solely on early EM, should focus on the subpopulation of patients who present with neurologic or other disseminated symptoms."

Page A-94:

"In a study of 60 U.S. patients with neuroborreliosis (16 with early and 44 with late neuroborreliosis), the sensitivity of PCR in CSF was 38% in early and 25% in late neuroborreliosis, and an inverse correlation was found between duration of antimicrobial treatment and PCR results (Nocton et al., 1996)."

So clearly, PCR isn't so great at detecting neuroborreliosis in early or late stage infection.

Page 7-16

"With respect to pathophysiology, Borrelia act directly and can invade neural cells in vitro (Livengood and Gilmore, 2006); there are also indirect actions, such as the induction of local cytotoxins or inflammatory mediators (reviewed in Fallon et al., 2010). European studies show that pro-inflammatory cytokines are increased, and chemokines, excitotoxin, and quinolinic acid are increased in patients with neuroborreliosis (Weller et al., 1991; Halperin and Heyes, 1992; Widhe et al., 2004; Rupprecht et al., 2005)."

(An aside: For all of those readers getting excited about the excitotoxin, it's not produced by the Lyme Borrelia spirochete - it's produced by your own body. If you're thinking about detoxing that, I'll be discussing it in a different post.)

A discussion item on this was noted at some point during the workshop:

"...Dumler noted that large-scale human clinical studies that have sufficient statistical power are needed. As discussed by previous panelists, such studies would allow the acquisition of large numbers of subjects and potentially bring together all of the involved communities — patients,advocate groups, physicians, academicians—to address research uncertainties on a large scale. These clinical trials for tick-borne diseases could easily be assimilated into modern high-throughput methods that may make whole genome surveys feasible.There would need to be some discussion on how many patients would be needed for a single-nucleotide polymorphism (SNP) analysis for neuroborreliosis. A large-scale clinical study would be intense and difficult, but it would rely on the communities coming together. These clinical trial groups could provide critical corroborated subjects and a biorepository of samples for pathogenesis studies. Within the group, one could create and validate the next generation of diagnostics. It would also provide a critical structure for the assessment of the new diagnostics, clinical interventions, and therapeutics.

Here's one action item: Organize a large-scale human clinical study involving single-nucleotide polymorphism (SNP) analysis.

But is this the most relevant test for people who are ill and have persistent symptoms? I wish Dumler would outline the process for patients as to what he sees in the future for patient treatment using this data.

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0 Exercise: A Better Lyme Disease Case Defintion

CDC painting by numbers: The numbers
need to represent reality - actual cases are much
higher than those reported to the CDC.  

Since many patients do not like the case surveillance definition for Lyme disease, I have an exercise for my readers.

I'm going to provide you with a case definition for Lyme Disease, and see what you have to say about it.

Do you think it is better than the current CDC case definition? Or worse? Why or why not?

What do you think needs to change, and how would you change it?

What do you think is missing? What should be added?



Please share your view in comments - I want to see what people say about this provided definition first and then see if we can collectively rewrite a better one.

Lyme Disease

Clinical description

A systemic, tick-borne disease with protean manifestations, including dermatologic, rheumatologic, neurologic, and cardiac abnormalities. The best clinical marker for the disease is the initial skin lesion, erthyma migrans, that occurs among 60-80% of patients.

Clinical case definition

• Erythema migrans, or
• At least one late manifestation, as defined below, and laboratory confirmation of infection

Laboratory criteria for diagnosis

• Isolation of Borrelia burgdorferi from clinical specimen, or
• Demonstration of diagnostic levels of IgM and IgG antibodies to the spirochete in serum or CSF, or
• Significant change in IgM or IgG antibody response to B. burgdorferi in paired acute and convalescent phase serum samples

Case classification: a case that meets one of the clinical case definitions above

Comment

This surveillance case definition was developed for national reporting of Lyme disease; it is not appropriate for clinical diagnosis.

Definition of terms used in the clinical description and case definition:

A. Erythema migrans (EM)

For purposes of surveillance, EM is defined as a skin lesion that typically begins as a red macule or papule and expands over a period of days to weeks to form a large round lesion, often with partial central clearing. A solitary lesion must reach at least 5 cm in size. Secondary lesions may also occur. Annular erythematous lesions occurring within several hours of a tick bite represent hypersensitivity reactions and do not qualify as EM. For most patients, the expanding EM lesion is accompanied by other acute symptoms, particularly fatigue, fever, headache, mild stiff neck, arthralgia, or myalgia. These symptoms are typically intermittent. The diagnosis of EM must be made by a physician. Laboratory confirmation is recommended for persons with no known exposure.

B. Late manifestations

Late manifestations include any of the following when an alternate explanation is not found:

Musculoskeletal system

Recurrent, brief attacks (weeks or months) of objective joint swelling in one or a few joints, sometimes followed by chronic arthritis in one or a few joints. Manifestations not considered as criteria for diagnosis include chronic progressive arthritis not preceded by brief attacks and chronic symmetrical polyarthritis. Additionally, arthralgia, myalgia, or fibromyalgia syndromes alone are not criteria for musculoskeletal involvement.

Nervous system

Any of the following, alone or in combination:

Lymphocytic meningitis; cranial neuritis, particularly facial palsy (may be bilateral); radiculoneuropathy; or rarely, encephalomyelitis. Encephalomyelitis must be confirmed by showing antibody production against B. burgdorferi in the cerebrospinal fluid (CSF), demonstrated by a higher titer of antibody in CSF than in serum. Headache, fatigue, paresthesia, or mild stiff neck alone are not criteria for neurologic involvement.

Cardiovascular system

Acute onset, high-grade (2nd or 3rd degree) atrioventricular conduction defects that resolve in days to weeks and are sometimes associated with myocarditis. Palpitations, bradycardia, bundle branch block, or myocarditis alone are not criteria for cardiovascular involvement.

C. Exposure

Exposure is defined as having been in wooded, brushy, or grassy areas (potential tick habitats) in a county in which Lyme disease is endemic no more than 30 days before onset of EM. A history of tick bite is NOT required.

D. Disease endemic to county


A county in which Lyme disease is endemic is one in which at least two definite cases have been previously acquired or in which a known tick vector has been shown to be infected with B. burgdorferi

E. Laboratory confirmation

As noted above, laboratory confirmation of infection with B. burgdorferi is established when a laboratory isolates the spirochete from tissue or body fluid, detects diagnostic levels of IgM or IgG antibodies to the spirochete in serum or CSF, or detects a significant change in antibody levels in paired acute and convalescent phase serum samples. States may determine the criteria for laboratory confirmation and diagnostic levels of antibody. Syphilis and other known causes of biologic false-positive serologic test results should be excluded when laboratory confirmation has been based on serologic testing alone.


Well, what do you think? What works? What needs rewriting and why?

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0 Shor Study on CFS and Lyme disease

This comes through as what seems to be an early release that can be viewed through Dr. Shor's website:

http://www.iacfsme.org/BULLETINWINTER2011/Winter2011ShorCFSinLyme109123/tabid/458/Default.aspx

ABSTRACT

Background

Chronic fatigue syndrome is a diagnosis of exclusion for which there are no markers. Lyme disease is the most common vector borne illness in the United States for which chronic fatigue is a frequent clinical manifestation. Intervention of patients with Lyme disease with appropriately directed antimicrobials has been associated with improved outcomes.

Results

Of the total 210 included in the analysis, 209 or 99% were felt to represent a high likelihood of “seronegative Lyme disease.” Initiating various antimicrobial regimen, involved at least a 50% improvement in clinical status in 130 or 62%. Although not achieving the 50% threshold according to the criteria discussed, another 55 patients subjectively identified a beneficial clinical response to antimicrobials, representing a total of 188 or 88% of the total identified as having a high potential for seronegative Lyme disease.

Conclusions

A potentially substantial proportion of patients with what would otherwise be consistent with internationally case defined CFS in a Lyme endemic environment actually have a perpetuation of their symptoms driven by a persistent infection by Borrelia burgdorferi. By treating this cohort with appropriately directed antimicrobials, we have the ability to improve outcomes.

More available at the above link.

Comments (briefly now, as I am headed out the door - may comment more later):

Kudos to Samuel Shor and his team for this research. Thank you for including potential limitations, future directions for research, and disclosures in your report.

Additional studies on this patient group need to be conducted, and I would like to see more such studies as these conducted and shared with the Lyme patient community, even if they are only retrospective and not controlled studies...

Use the clinical data you have on all of us, anonymously - at least we have some idea what sort of outcomes you are seeing?

This looks like the way to go, Dr. Shor: 

"Obtain microarray analysis for Borrelia burgdorferi on the “seronegative” Lyme patients. Perform a prospective randomized placebo controlled trial for which a protocol and  IRB are already in place and funding being pursued."

Yes. Thank you. More sir, may we have another?

It's a start...

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0 Books: Mini Review on "Bull's Eye"

I've been reading bits and pieces from Bull's Eye: Unraveling The Medical Mystery Of Lyme Disease.

Someone told me it may be a frustrating read, so I was prepared for that - but it hasn't been that difficult to get through, actually, once you get past the fact that the author can write about Alan Steere more matter-of-factly than anyone I know.

That's one of the main criticisms I've heard other Lyme patients who have read it have about the book - but after reading a huge chunk of it, I'd have to say that it is more balanced about discussing the controversy than I thought it would be and that much of it focuses on things other than Steere: the history of Lyme disease in Connecticut, history of Borrelia in Europe, general research on Borrelia burgdorferi, epidemiology, difficulties in diagnosing Lyme disease, problems with serological testing, the Dearborn criteria, coinfections, shortcomings in the IDSA view of Lyme disease, the Ed Masters story, experimental treatments, genomes, vaccines, and legal issues.

And it's an easy read, for what it's worth - for the most part, people who aren't knowledgeable about Lyme disease or its controversy can jump right in and begin getting an idea of the big picture pretty quickly.

One of the things that makes it readable is the author, Jonathan Edlow, MD,  can outline procedures using metaphors and analogies in a basic way so that those reading this kind of material for the first time can 'get it', and I've found  the Ambiguity in the Lab chapter to be one of more interesting reads because of how it was written.

In the book, the author describes for the reader how Western blot tests are done for Lyme disease:

"Like the ELISA, the Western blot tests for antibodies, but it allows the laboratory to find precisely which antigens a patient's blood contains antibodies to - not just whether there are any kinds of anti-borrelial antibodies. 

First, the B. burgdorferi is put into a detergent to break up all its proteins. Each of these proteins has different sizes and each has a slight electrical charge. These proteins (antigens) are placed on a gel to which an electrical current is applied. The proteins then migrate across the gel, which is full of nooks and crannies, rather like an English muffin. Because the proteins have different sizes, they move through the gel at different rates, the larger ones moving more slowly than the smaller ones. After a certain amount of time migrating with the electrical current across the gel, the proteins from the B. burgdorferi have traveled different distances. 

Imagine a massive jungle gym with evenly spaced bars going every which way. A group of people who are four to seven feet tall are instructed to start at one end of the jungle gym and travel through the latticework as far as they can in five minutes toward the other side. In this group of people, ten are exactly four feet tall; ten are exactly four feet, six inches tall; ten are five feet tall, and so on - such that there are seven groups of ten people who are all the same height. 

Since the spaces between the bars of the jungle gym are uniform, the smaller people will be able to travel faster across this jungle than the larger, heavier people. When the five minutes are up, the larger people will be closer to the start and the smaller closer to the finish. Assuming that the participants are equal in their abilities, at the end of the five minutes, the seven groups will settle out in seven distinct regions of the jungle gym. If one were to take a picture of the apparatus at the end of the five minutes, it would show seven bands - clusters of people of the same height - interspersed with bare areas of the jungle gym devoid of anyone.

This is what happens to the proteins from borrelia in the Western blot. They migrate across the gel, through the network of obstructions, at different rates on the basis of their molecular weights. After a specified period of time, the proteins are clustered on the gel at specific regions. They are then transferred (blotted) onto a special kind of white membrane. Specific antibodies (attached to a dye and so that they can be seen) will bind to specific borrelial proteins, and colored bands will appear on the white membrane."

Edlow explains other processes and definitions by using metaphor and analogy throughout the book, making it easier to visualize them. He uses the idea of a machine to sort different kinds of fruit to explain what sensitivity and specificity mean in terms of blood tests detecting infection, and uses different models and makes of cars to help describe cross-reactivity in testing. The way he puts things makes it easier for people with no prior knowledge of Lyme disease and testing to understand certain concepts that they can later build on.

There are other reasons I like this book, and a few reasons I don't, but I wanted to take time aside to share this one aspect.

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0 Paper On ELISA And Immunoblot Differences

The Lyme patient community has been saying all along that blood tests for Lyme Disease are inaccurate and vary in specificity and sensitivity for some time.

This recent paper is more data in support of this complaint.

Full text version: http://www.springerlink.com/content/w64170894v08654g/fulltext.html

Eur J Clin Microbiol Infect Dis. 2011 Jan 27; [Epub ahead of print]

C. W. Ang1 Contact Information, D. W. Notermans2, M. Hommes1, A. M. Simoons-Smit1 and T. Herremans2
(1) VUMC, Amsterdam, The Netherlands
(2) Centre for Infectious Disease Control Netherlands, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands
Received: 21 July 2010 Accepted: 1 January 2011 Published online: 27 January 2011

Large differences between test strategies for the detection of anti-Borrelia antibodies are revealed by comparing eight ELISAs and five immunoblots.

ABSTRACT

We investigated the influence of assay choice on the results in a two-tier testing algorithm for the detection of anti-Borrelia antibodies.

Eighty-nine serum samples from clinically well-defined patients were tested in eight different enzyme-linked immunosorbent assay (ELISA) systems based on whole-cell antigens, whole-cell antigens supplemented with VlsE and assays using exclusively recombinant proteins.

A subset of samples was tested in five immunoblots: one whole-cell blot, one whole-cell blot supplemented with VlsE and three recombinant blots.

The number of IgM- and/or IgG-positive ELISA results in the group of patients suspected of Borrelia infection ranged from 34 to 59%.

The percentage of positives in cross-reactivity controls ranged from 0 to 38%.

Comparison of immunoblots yielded large differences in inter-test agreement and showed, at best, a moderate agreement between tests.

Remarkably, some immunoblots gave positive results in samples that had been tested negative by all eight ELISAs.

The percentage of positive blots following a positive ELISA result depended heavily on the choice of ELISA-immunoblot combination.

We conclude that the assays used to detect anti-Borrelia antibodies have widely divergent sensitivity and specificity.

The choice of ELISA-immunoblot combination severely influences the number of positive results, making the exchange of test results between laboratories with different methodologies hazardous.

---

It would be good to see an analysis like this on all North American ELISA and immunoblot tests - this paper focuses on European tests. 


The full text for this paper showed that not only does two-tier testing miss positive cases, but that  specificity between immunoblots varies greatly and can miss positive cases. The implication is that using some immunoblot tests to confirm positive ELISA tests may be a flawed approach due to differences between immunoblots. 


The discussion section states that, "Theoretically, the use of recombinant antigens should lead to increased specificity and, possibly, increased sensitivity as well. This does not seem to be true for the currently available ELISAs and immunoblots for the detection of anti-Borrelia antibodies. We could not find a clear relationship between the fraction of positive tests, the specificity and the nature of the antigen used for the serological tests. ELISAs using sonicated whole-cell antigens can be sensitive and specific, while recombinant ELISAs may lack specificity. Therefore, manufacturer claims for the superior performance of assays using recombinant antigens for the detection of Borrelia antibodies must be interpreted with caution."

Problems in detection occur with both the first tier and second tier of testing, and results are further  affected by how early in the infection the patient is tested and whether or not the patient has had any antibiotics recently.


This seems to confirm the need for more awareness of how sensitive and specific each lab's tests are at each tier, and to rely more on clinical diagnosis and patient history to determine treatment.

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