Diagnosis
A more accurate diagnosis: Lyme Disease
A much more relevant diagnosis was found much later, in fact 9 years after my son’s initial regression: Lyme’s. This diagnosis was not found by our NHS consultants, as I said earlier, as far as they were concerned, our case was closed. In despair, I had returned to France, hoping that I would get more answers from a series of lab tests conducted by Prof. Montagnier, following the advice of Dr. Skorupka. Other tests were made on the same day, as well as an electro-encephalogram (EEG). The other tests did not show any particular relevant information, though the EEG, showed a classic low frequency wave (theta activity 4-7Hz, regular, widely spread over the whole scalp area, bilateral and symetrical), without epileptic activity which was interpreted in a different light than the one I was used to in the UK. I will discuss this in another section on this web site.
The presence of Borrelia burgdorferi was initially detected by Polymerase Chain Reaction (PCR). Now, let me tell you a little more about this method of detection as I have come to understand that there is a lot of misunderstanding about what this technique is about, even from medical doctors themselves. For those who want to skip the explanation on PCR skip to Confirmation of the initial diagnosis below.
PCR allows the detection of genomic material, either RNA (with a prior step of reverse transcription) or DNA, using a set of primers specific to the target DNA sequence. Primers are short oligonucleotide sequences of 10-25 base pairs that will specifically hybridize (bind) to the target DNA sequence, by complementary base pairing. The primers are then extended by the DNA polymerase in the presence of nucleotides. This cycle is repeated several times, 30-50 times depending on the conditions, in order to generate sufficient amplification for detection. The band is then analysed on a gel by electrophoresis to compare its size to DNA markers. The size is initially used as way of identifying the product and confirming its identity, but often the amplified product is further analysed for example by enzyme restriction digest (using an enzymes that is expected to cut the target at specific sequences leading to the expected digestion products according to their size), or by cloning and sequencing.
There are several potential artefacts to a PCR. One is that the product obtained can be a contamination, leading to a false positive result. This is in fact something that is extremely common in labs. The reason is that a PCR is really sensitive and can pick up minutes amounts of DNA. If the target DNA is around in the lab, which is the case as prior PCR have been performed, either as ongoing testing or to establish the assay, the chances are very high that other reaction tubes could get contaminated. Good laboratory practice requires that absolute care is being taken when performing a PCR assay. Generally you work with gloves, filtered tips (to avoid contamination from the pipette), under a hood, with absolutely clean reagents and material. You also use water as control (No DNA control), and it should remain totally clear of any amplification if there is no contamination. With practice, you can get a very good sense of whether or not the results come from a contamination or are genuine. Repeating the assay and finding the same pattern or positive and negative products should also confirm that the results. In short, if no proper care is taken, a PCR can give rise to false positive results.
The second type of problem is that the PCR can be negative, either because the assay is not working reliably (primers or detection of very low target number), or because the biological sample from which the assay is ran does not contain the target. We have to keep this in mind, that the body is complex. Often we only have access to blood sample to run a PCR, if the blood does not contain any material, because say for example the pathogen is not found in the blood, then the PCR will be negative. Regarding the detection of Lyme’s pathogen, it has been estimated that only 25% of the cases (who have developed a classic erythema migrans rash together with the symptoms of the disease) are positive by PCR. This indicates that indeed the spirochete genomic material is rarely present in blood sample, so a negative PCR result does not rule out an infection.
A further clarification I wish to make is that even though PCR can detect trace amounts of genomic material, it does not mean that the result is benign in terms of its significance. The genomic material of Borrelia burgdorferi should not be present in people, full stop. If it is, even if it is at low level, it indicates an earlier infection. And this is sufficient to demonstrate Lyme disease.
Now having explained all of this, the results we initially obtained indicated a positive result, which was confirmed 3 times. The PCR product was sequenced and its identity: Borrelia burgdorferi was confirmed by its sequence.
I felt none the less that it was important to confirm this result with further independent testing.
Confirmation of the initial diagnosis
The confirmation was made using a test called Borrelia burgdorferi (Bb) Elispot LTT. Basically a blood sample was placed in presence of Bb full antigen, a Bb peptide mix or Bb LFA-1 (LFA-1 stands for Leukocyte function-associated antigen 1- and it refers to the immunodominant epitote of Outer Surface Protein A (Ospa). This epitope is similar to human LFA-1, which leads to possible auto-immune reaction). This Elispot measures the release of interferon gamma by reacative lymphocytes. It is a test of T-cellulal immune response. If the organism has not been in prior contact with a given antigen, there will be no reactivity. These tests were conducted at the Borreliose Centrum Augsburg in Germany.
The test showed:
Borrelia burgdorferi Elispot LTT
Borrelia Full Antigen + 4 SI < 2
Borrelia Peptide Mix + 3 SI < 2
Borrelia LFA-1 2 SI < 2
The results of the Elispot-Lymphocyte-Transformation-Tests are an indication for an actual cellular activity against Borrelia burgdorferi.
There was also measurement of IgM and IgG against Bb by immunoblot. This is a test that is known to frequently be negative because the host immune system is not easily capable of developing an humoral (antibody-mediated) immune response against this pathogen. The results were indeed negative.
Borrelia burgdorferi antibodies (immunoblot)
Borrelia-Blot-IgG-antibodies negative negative|
Borrelia-Blot-IgM-antibodies negative negative
Further, a CD 57 (Natural Killer cell- type 1) flow cytometry was performed and showed the typical immune suppression of NK-1 (CD57) cell count seen in Lyme disease.
CD 57 Flow Cytometry
Leucocytes 6.41 /ul 2.6-10.0
Peripheral Lymphocytes 49.1 % 18.0-51.0
Lymphocytes 3147 /μl 468-5100
Natural killer cells 9.54 % 6-29
Natural killer cells 300 /μl 60-700
CD 57 positive NK-cells 3.02 % 2-77
CD 57 positive NK-cells – 95 /μl 100-360
The CD57-cell-count is an indication for a chronic immune-suppressive situation caused by Borrelia burgdorferi.
Other co-infections were also screened for as a tick bite can also transmit other pathogen, Ehrlichia was borderline positive whilst none of the other pathogens were found.
Ehrlichia Elispot LTT
Ehrlichia-Elispot LTT 2 SI < 2
The result of the Elispot-Lymphocyte-Transformation-Test is an indication for a borderline actual cellular activity against Ehrlichia.
Yersinia antibodies
Yersinia-IgA-antibodies (immunoblot) negative negative
Yersinia-IgG-antibodies (immunoblot) negative negative
The specific Yersinia-IgA- and –IgG-antibodies are no indication for a humoral immuneresponse against Yersinia spp.
Bartonella antibodies
Bartonella henselae-IgG-IFT < 1:320 Titer < 1:320
Bartonella quintana-IgG-IFT < 1:320 Titer < 1:320
Serological no evidence for an infection with Bartonella henselae or Bartonella quintana.
Babesia divergens antibodies
Babesia divergens-IgG-antibodies < 1:16 < 1:16
No serological evidence for an infection with Babesia divergens.
Toxoplasma gondii antibodies
Toxoplasma gondii-IgG-antibodies 0.3 IE/ml < 1.60
Toxoplasma gondii-IgM-antibodies negative negative
No serological evidence for an infection with Toxoplasma gondii.
Anaplasma phagocytophilum antibodies
Anaplasma phagocytophilum < 1:64 < 1:64
IgG-antibodies
Anaplasma phagocytophilum < 1:20 < 1:20
IgM-antibodies
No serological evidence for an infection with Anaplasma.
The presence of auto-antibodies was also assessed. No antinuclear antibodies were detected.
ANA (EIA) 0.07 Ratio < 1.0
Included are auto-antibodies (IgG) against the following antigens: Chromatin (dsDNA,
Histones), Sm/RNP, SS-A, SS-B, Scl-70, Centromer, PCNA, Jo-1, Mitochondria (M-2),
Ribosomes, nuclear extracts.
There is no evidence for antinuclear antibodies.
Together with the symptoms presentation, these results indicate conclusively a Lyme diagnosis.
I would like to take this opportunity to thank Dr. Skorupka, Prof Montagnier, Dr. Nicolaus, Dr. Schwarzbach and the wonder staff at the BCA.
