Now moving on to laboratory criteria. I did have a slide that I am sorry I couldn’t transpose to Powerpoint presentation but at the end of one of our sessions while I was at the University of Connecticut and it was a multi-disciplinary clinic in which we had several physicians who did not agree with each other but nonetheless we agreed to study these people. And we had signs posted up like a score for a skating competition. One said at the end of the presentation one held up ‘LYME’ another held up ‘NO LYME’ another one held up a sign saying ‘IT JUST DOESN’T MATTER’ and another one held up a sign saying ‘I WASN’T LISTENING’. And it just shows you the frustration that is within the physicians as well as the patients when you have multiple symptoms that are difficult to ferret through.
So you would think the laboratory would be our saviour. Go to the laboratory. That is what you do to figure out what is wrong with somebody. And the answer is, unfortunately, we have a ways to go. And what are the problems here? I think Dr Owen reflected some of them but the first test, just because that is what we have done over the years, is to go to serology. Let’s get our immune system, our antibody system, our circulating antibody system, to tell us: have we been exposed to the agent. Yes or No. And when this was first done and we said let’s go through, we’re good at making enzyme -immuno assays. We’re good, we get these 96 well plates running through the machines. We’ll grind up the organisms. We’ll put them on the plate. We’ll get the patient’s serum. We have the anti-anti serum. It’s a nice colour, we can measure it on the machine and out it comes.
And so when this was done and the results were published both of the ELISAs and the subsequent Western Blots, there was very poor inter-laboratory co-ordination and this was published in the mid-90s and there were a variety of laboratories; University of Connecticut was one of these and they were sent blind specimens – and these were all cases of proven Lyme Disease. What we call Late Lyme Disease. Dr Steere liked to call Early disseminated with Stage 1, 2 and 3 first and then it became clear that there weren’t separate stages just a continuum of early disseminated and late as he liked to call it as apposed to chronic. And those were patients with big swollen knees with lots of robust IgG responses that the laboratory sensitivity by the titre was 50 to 55% and with the blots that he went through and published this Western Blot information that when you look at the individual bands and on Western Blot basically it is a technique of separating out the various proteins of whatever it is you are looking out. In HIV you separate out their proteins, in Lyme you separate out their proteins, you pass an electric field through a gel and then these are the various proteins and more.
Some of the outer surface proteins - basically what you are measuring of the Lyme bacteria, the Lyme Borrelia, and then analysing how many of these are shown, that is, what antibody reactions to these are shown, in various patients groups. And in the Journal of Infectious Disease report in 1994, Dressler and Steere, you could see that if you had reactions against what you consider highly specific proteins, that is what they looked at, you only needed one reaction. But somehow this got convoluted so that when they met in Dearborn in Michigan in 1994 they declared that you needed to have 5 or more reactions by IgG or 2 reactions by IgM in order to have a positive test. And for the life of me I have not been able to figure that out because in their actual study - let alone that they did not look at any chronic patients, they did not look at any late Lyme Disease patients really other than the big swollen knee, so we have no information from them. The information as I have presented it – there have been a few people from Europe who have said that it isn’t that way for the Late Lyme patient and I will try to explain why I think this is the case.
Question from floor:
Q You mean they looked at Lyme Arthritis patients rather than Neuro-borreliosis patients?
A Yes, they said they looked, and if you look at the numbers of the meningitis what they called meninlo- encephalopathy it is like 5, 10…
Q I understand that Lyme Arthritis people get far more bands than others.
A Yes, they do, they have robust responses and they actually do quite well and there may be an important part of why the chronic Lyme patient who was immune responsive before does worse.
Q But the neurological person tends to get a lot of bands above and between 60 and 93?
A There may be and this now raises the issue of whether (inaudible interruption) the European strains are any different than the American strains and I think this is still an open question as with the old Rheumatic Fever and Rheumatic Heart Disease - that licked heart and bit the joints or bit the heart and licked the joints. Do you have different strains that are more rheumatogenic and other strains that are more neurotrophic and I keep that open but I think we have appreciated that in the United States because Rheumatologists were looking at this disease that they emphasised the rheumatologic manifestations and did not seem to appreciate the neurologic manifestations whereas in Europe it was a neuro-Borrelia or neurologic disease sort of syndrome and neurologic disease to begin with, and now you realise that a lot of the European patients do have arthralgias/ myalgias. In fact, in chronic Lyme Disease patients – I didn’t show you that separately – arthritis itself, which by definition is a swelling, as apposed to arthralgias which is an aching without any swelling, occurs in about 25% of the chronic Lyme patients so that means 75% of the patients have joint pains mostly pains without any swelling. And we have swelling in the chronic patient. You can have the knee involved but atypically it is also other places, the elbow or a finger or the wrist and it makes it very difficult as you go through the differential diagnosis of each of these disorders.
But what we have been finding is that when you look at the Western Blot this is a more sensitive technique. To look at it, it is still not a perfect technique because it only uses one dilution. You take a particular amount of the patient’s serum and you dilute it one hundred fold and you use that particular dilution against this strip of paper that contains all of the antigens on the paper and ask the question: is there antibody that recognises any of these specific proteins which numbers are according to size, molecular weight as it is called, chemical weight, in size and if you see a reaction, you say it has seen this particular protein and if you don’t you then you don’t have that evidence for it.
But looking at this, what we have been finding is that patients with the chronic phase have IgM reactivity, preferentially IgG reaction. Now some of this has been then thrown out as saying well this has to be a false positive. There is no such thing as a false positive blot, I mean, you either have the reaction or you don’t have the reaction. The interpretation is something else and you can argue about its significance and I will show you some information about that and I will return to the DNA culture in the spinal fluid. Let me just show you what we found in our patients and this is in the first tetracycline paper that I published in 1997 that here is the ELISA being either positive or negative and Western Blot being positive or negative and in this case my definition was 2 or more reactions, one of which had to be specific either IgM or IgG, and according to that when you have an ELISA that is positive, you only have a negative blot in 1% of people. When you have an ELISA that’s negative, you have a Western Blot that is positive in about 50% of people. So you are missing half the people if you just stop at an ELISA that is being negative. And both are negative in about 18-20% of people. So that means, well wait a minute, that must mean that they don’t have Lyme Disease or it must mean the test is not as sensitive as it might be because in some of these patients they have had other compelling evidence being a tick bite, the erythema migrans rash partially treated, some PCR DNA evidence.
Nonetheless, one is, I think, authorised to question whether these people then do or do not have Lyme Disease. One of our later studies that appeared in a recent publication of Medical Science Monitor a year ago on macrolide treatment of chronic Lyme Disease is showing there are more details. Here you have ELISA positivity or negativity in this column and this is IgM or IgG reactivity and what you see just jumping here is that when the ELISA is positive the blot is negative in only 6% of people. When the ELISA is negative, the blot can be negative in up to 33% of patients.
So, even with the best of intentions, you come out with a population which appears to fit the criteria of chronic Lyme Disease who are sero-negative a third of the time. What do you do with those patients? Well, in the beginning, when I first started to practice, I dealt with the sero-positive people and then I began to enroll, if you will, the sero-negative people and it became clear to me afterwards that either I was stupid or I could not tell the difference between the two of them because the reactions did not depend upon their Western Blot reactivity. They seemed to react more because of the duration of the disease and the type of treatment we gave them than according to this blot. But when you look again a little closer, you see that IgM and IgG reactivity is common in these patients together but you will get some isolated IgM reactivity especially in this ELISA negative group here and these are reactions to 23kD protein which is the outer surface ‘C’ as it is called, that a group in Austria that are trying to generate a vaccine and a group in Maryland is also trying to get a vaccine against this outer surface protein ‘C’ so we know it is a highly specific protein.
Before I get into the Imaging Studies, I am going to say a few words about the other techniques. Direct culture has been very difficult. Why has it been difficult? You can actually culture the organism. You can culture it from skin, from the erythema migrans and in a SmithKlineBeecham study of a vaccine where they found that half the patients had atypical rashes (so it was one value of that study showed that erythyma migrans, bull’s eye rashes, were present in about 50%, maybe less, other rashes 50-60%. ) When they cultured those rashes, they were able to culture the bacteria.
I hope they still have them because we would like to do some gene studies on antibiotic resistance genes, for example, because we haven’t had any antibiotic studies of Borrelia sensitivity since the early 80s and that needs to be re-done, if you will, from fresh isolates just to see if that has changed. Is that a factor in antibiotic responsiveness or lack thereof in on-going Lyme Disease patients. So the culture, thereafter, except from an isolated report from an eye or from wherever, cultures have not been very rewarding even in the animal. The primate studies from Tilain or some of the elegant dog studies done at Cornell by Strobinger, he was not able to culture the bacteria back. What he did find was in some of his models was that PCR DNA which disappeared with initial antibiotic treatment. reappeared over a series of 5 to 6 months suggesting that there was relapsing disease.
PCR DNA is a very sensitive technique in the sense of, and very specific, to the probe, the sequence, that you design it with. The problem that we have had with PCR DNA in medical practice in infectious diseases is that the technique is too sensitive in the sense why don’t we just do blood cultures and do PCR DNA on them? Why is it that we have to wait for culturing them just to get the gene material and I thought 10 years ago or more that this would have been what would have happened and I think what you will find is that the technique is so sensitive that you are probably picking up transient asymptomatic bacteriaemias. Or in the case of Legionella Disease, or Streptococcal disease or Epstein Barr Disease, you find the organism, or evidence of the organism, the DNA, for months afterwards. Does that tell you that you have carriage of the organism? Probably it does.
With Lyme Disease, I think it had been hoped, that this would now give us a more sensitive way of telling whether you have Lyme Disease. So they looked in serum, they looked in whole blood actually. Because if you are going to find the DNA, it is going to be in the scavenger cells, in the white bloodcells, it is not going to be floating free as HIV RNA can be found, or Hepatitis can be found, and the problem has been that it has been rare to find any PCR DNA positivity in blood, spinal fluid or urine.
It is there, and so now comes the issue, how do you interpret this? Is it a real thing which means that a person has been exposed. It certainly is not a marker of active disease in that the PCR DNA test does not tell you if you have an active infection. It just tells you that the DNA is there. It could have been there 20 years ago, it could have been there yesterday. You might wish to assume that it is a marker of active disease but it isn’t in the same way we have the viral load for hepatitis and HIV. We need a messenger or RNA test or we need a more active, a probe of active infection, and we don’t have it yet. And in the spinal fluid, it was so disappointing that after the first several hundred people when I was getting back negative after negative, I stopped doing that. And even though we had a paper that bounced around and couldn’t get published that PCR DNA positivity was sero-negativity; the argument came back, well, how can you be sure even in the best laboratory that it isn’t a contaminent.
And I think that is a real concern. Persing has expressed this concern ad nauseum but it is true, when we site visited labs in order to select labs to confirm our mycoplasma DNA results for Gulf War Illness, it became clear when you went into the laboratory: A technician is a human being. They store their lunch in the refrigerator that they shouldn’t be storing it in. They leave the tubes open when tubes should not be left open. There are other materials. So, they put the right controls in the racks. They have positive controls, they have negative controls and they report out a PCR DNA tests. I do not know if it is positive or not. I can use it. I can use it politically or in a particular legal case and say this means the person had Lyme Disease but scientifically I don’t know what to make of it.
Do I do PCR DNA tests? Sometimes I do. But they are not as reliable as I would like them to be. With Herpes encephalitis, for example, PCR DNA test has replaced culture and antigen detection in the spinal fluid and it is positive for about 2 weeks and then it disappears. What does that tell us? It tells us that we haven’t eradicated the Herpes virus, it is still there. And I think what the Lyme PCR DNA tests and what the antibody tests are telling us, I think what they are telling me, is that it is not where we are looking. It is like your typical story, you lose your dime in the dark, and where do you look, under the lamppost. It is elsewhere, it is probably inside cells. And this disease is such an indolent disease. It is not very surprising is it if you think about it from that point. It is not going to be an aggressive, fulminant life-destroying disease like a cancer or Hepatitis disease that is running rampant. So it is not surprising to me that it may stay intra-cellular for the rest of its life and somehow cause havoc on the individual nerve cells or the ganglia or whatever part of the brain that they have been probing for.
Whether we are ever going to be successful in finding a better marker or a better scent of the bacterium remains to be seen. But I am optimistic that we have to be able to find a metabolite of some sort that somehow comes out into the circulation, and perhaps into the urine, perhaps into the spinal fluid that will make a better test. We know that antibody tests are not good tests for ongoing disease. And whoever follows an infectious disease anymore with an antibody to tell you if you are over and, when you do antibodies for Herpes disease, to see you have Herpes as a cause of your disease you are deluding yourself because all that tells you is that you have been exposed to the antigen that may or may not have anything to do with the illness in question and I think that our antibody tests are all that we basically have and we need to have more effort devoted towards study. We have the technology, we can do MMR on bacterial filtrates metabolite to see if there is a unique form and then begin to study it. But there is very little interest at the national levels of funding research and it is one of the purposes of meetings like this is to get people together and begin organise into more effective groups to get government research to have a more serious approach to this disease.
Question from floor:
Q.Can I just ask you a very quick question? Surely, if we can actually see the bacteria under the microscope as already shown us then could we not just then use the PCR to confirm what we have seen?
A. We are going to get into a controversial area here but the question is that there are some laboratories who are saying well we see the bacteria and when Phillips and Macknin first looked at their studies, and I know Steve Phillips very well, and he sent me the paper at the time, and I said, Steve you have to do the DNA studies on this to correlate it. Seeing it is not enough because I don’t know what you see. I think you know you can question what you see yourself and you know, and plans are to try and validate what people see and it needs to be done with specific monoclonal antibodies or DNA tests in a blinded fashion and validated. I think if it was that easy to tell you the truth, and to jump ahead with my opinion, I think those DNA tests in the circulation would be positive all the time. There probably is transient spirochaetemia but I don’t know that. I don’t know how much spirochaetemia exists after the initial infective episode.
Q Are you saying that the load in serum is so low that the DNA can’t be measured? That it is really in the tissues and the PCR can’t get at that?
A We are not measuring tissues and are we going to be biasing spinal nerve ganglia? I don’t think so. Yes, but may be there are other ways to do it, maybe peripheral neurons. We need a registry of tissues so that post mortem they can be examined for the location of all these forms that we are talking about. Certainly bacteria once they are dead inside cells they no longer have their spirochaetal long form they curl up like most other things. Whether you want to call this a cystic form or not it something to be debated later.
Let me get on. So I said about the culture and about the PCR DNA. I am hopeful there will be new advances. We certainly need to do research into better ways of monitoring the disease in addition to just asking the patient how you feel. Which I am comfortable with, I think that ultimately is the test.
What has happened with imaging studies. Well MRIs have been done and basically they are normal in 90-95% of people. This comes from extensive numbers of studies that we have done over the last 10 or 5 years or so and I am going to be publishing this. I have presented this in abstract form already. In which what happened was the first sign of abnormality was that you had these T2 signal hyperintensities that were routinely called MS in the early 90s and then they realised that Multiple Sclerosis is not the only disease that can give you these signals and that Lyme Disease can do it. And currently, I don’t know of any radiologist who’s looked at Lyme Disease MRIs and MS MRIs and can tell the difference from the next. So when you have this, it tells you you have this realisation this kind of scarring, the demyelisation present but it doesn’t tell you what’s causing it but it is compatible.
In contrast to that, the brain SPECT scan, was a single photon emission. Its like doing a bone scan but doing it of the brain. You see abnormalities in about 75% of patients. We have been doing these now for a number of years and the technique has improved so you have these two headed scopes and you can follow patients. This is not something that is abnormal one day and normal the next day. This was trivialised initially by some of what I call the ‘early experts’ who said that this is non-specific. I don’t want my brain to look like that and I don’t think that it is non-specific. And the good news is that it is reversible with good treatment and I think that, to me, is a strong note of optimism that we can do something with even long - standing chronic Lyme Disease. If we can reverse the SPECT scan changes, I think along with the clinical changes that we can see, that we are doing some good.
Here is a graph of some of the locations of the SPECT scan abnormalities. In the temporal lobe which is the cognitive area , the frontal area which would be the mood, poriatal is a mixed bag there. There can be tremors associated… a variety. Occipital and periventricular are very uncommon and there are some other areas: Basal ganglias, singular gyrus have been a few abnormalities of patients with Lyme Disease. And, as with other elements, it ranges from mild to moderate to severe. And, I don’t have one to show you here that with treatment it moves from the severe to the moderate to the mild and it disappears with successful treatment and it doesn’t disappear if the treatment is unsuccessful.
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