Falsified: Microscopy method is not useful for detecting Borrelia & Babesia

[duncan]

Understood.

What I'm saying is that what might seem like obvious logic may not hold true, i.e., just because you can see the buggers doesn't mean PCR will register similar success in even the same sample.

I do not know enough about microscopy to speak to its accuracy.

 

[lauluce]

I think his point was that if it was possible to spot it using a microscope, it would be so clear that PCR would be positive too. His conclusion is that this LM microscope method is a big scam.

He (dr. Aavitsland) is the former Deputy Director General of the Norwegian Institute of Public Health. He usually responds on Twitter:

https://twitter.com/i/web/status/702797328361590784

there is a guy who sells dark field microscopy of borrelia spirochetes as a diagnostic method, it is depicted on this video:

I can´t find the site of his organization, but I always suspected it was a SCAM, BEWARE!

 

[alicec]

The efficacy of PCR was gauged at times against culture-positive cases, and I don't think it did all that well. I think its range was 10 to 60%?

Well not as found in the review I referenced above. Here are a couple of quotes.

When skin biopsy specimens are first cultured and the culture
material is analyzed by PCR, the test sensitivity should be
higher because of the multiplication of organisms in culture. In
one such study in which a nested PCR targeting the ospA gene
was used, 8 of 10 patients with EM were positive; the interval
between the tick bite and the presentation of the patients
varied between 1 week and 3 months. The sensitivity of PCR
(80%) was considerably higher than that of culture (14%) or
serologic testing (77%) (116).

Recently, plasma from 76 patients with EM was analyzed in
a prospective, blinded study of PCR (51). Spirochetemia could
be documented in 14 patients (18.4%) by PCR (Table 4); only
4 (5.3%) were culture positive. Positive PCR results correlated
with clinical evidence of disseminated disease: 10 of 33 patients
with systemic symptoms (30.3%) were positive compared with
4 of 43 without such evidence (9.3%).

The sensitivity of culture is lower than that of PCR in all
studies. Even when PCR was performed on cultures of skin
biopsy specimens from patients with EM, culture was less sensitive
than PCR (161). After 2 weeks of culture, PCR was
reactive in 19 of 20 patients compared with 14 of 20 patients by
microscopic inspection.
No positive cultures have ever been found in CSF samples
from patients with late disease (93), and cultures from SF
succeeded in only a few patients with LA (123). Therefore, in
a patient with a negative culture and a positive PCR result, the
likelihood of a false-negative culture is much higher than that
of a false-positive PCR result.

 

[matsli]

@deleder2k

Why PCR is not a safe method
The lyme spirochete has developed strategies to escape the immune system. An example of this type of mechanism is antigenic variation. It has been defined as changes in structure or expression of antigenic proteins that occurs during infection at a frequency greater than the usual mutation rate.

Among the problems with Lyme disease is its variable outer protein patterns and that it is highly glycosylated proteins. The glycosylated proteins ensures that the antibodies can not be formed. It is the same type of defense that HIV uses. Of HIV envelope protein is highly glycosylated, so that there are no opportunities to find antigens in the housing, they are obscured by the sugar such that the antibodies prevented the retrieval höljantigenerna.

OspE proteins mislead
Spirochetes (lyme) can carry many similar plasmids with varying forms of OspE. In this way, Borrelia vary the OspE proteins that express on their surface and replace the plasmids with other Lyme disease bacteria. This may contribute to increased protection against the immune system.

Conclusion - PCR is not reliable
PCR has the problem that the different variants of Borrelia requires each it's own primer. You must know exactly what you're looking for, You can only get the answer to the question posed, not all the questions are not set.

For those of you who are thinking about ELISA
ELISA for Lyme disease have very low sensitivity according to several international studies and should not be used at all.

A safer method is Elispot (iSpot Lyme in the US)
Sweden has not yet approved the Lyme disease test Elispot. In principle Elispot counting the number of "gamma interferon spots" that T cells produce in Lyme disease.

A word about microscope by darkfield
In another study with darkfield microscopy. 8 patients with ME / CFS and 3 people without the diagnosis were suspected that they had Lyme disease. 10 of 11 had been tested for Lyme disease in healthcare. All had negative results. All were subjected to darkfield microscopy. In the film you can see that all have the spirochetes.

Read more here

 

[alicec]

PCR has the problem that the different variants of Borrelia requires each it's own primer.

That statement is so ludicrous that it casts doubt on everything else in the article.

The sequence of the entire genome of Borrelia and all of its 21 plasmids has been known for some time. As with other bacteria, there are highly conserved as well as hypervariable regions. Based on these sequences, PCR primers can and have been designed to amplify a wide range of DNA targets - all species, individual species, individual plasmids - it all depends what you want to study.

Various targets have been used to develop PCR assays for Borrelia in biological samples and you can find plenty of publications which compare the results and help define primer pairs suitable for robust assays. As far as I can see, none of them use the supposedly misleading OspE protein as quoted above - in other words this is a complete red herring.

 

[matsli]

@alicec

http://www.medscape.com/viewarticle/764501

 

[matsli]

PCR test - This test looks for the presence of the Lyme bacteria’s DNA in blood to confirm a diagnosis. Your blood sample may not have any Lyme DNA in it because when Borrelia is in its cyst-form, it rarely releases any. Also, people with Lyme disease have periods of time where they are more or less symptomatic or asymptomatic, which is reflective of the bacteria’s activities. The PCR test can come back positive or negative depending on the activity level of the infection.
http://www.tiredoflyme.com/4-reason...-even-if-a-person-truly-has-lyme-disease.html

 

[alicec]

PCR test - This test looks for the presence of the Lyme bacteria’s DNA in blood to confirm a diagnosis. Your blood sample may not have any Lyme DNA in it because when Borrelia is in its cyst-form, it rarely releases any. Also, people with Lyme disease have periods of time where they are more or less symptomatic or asymptomatic, which is reflective of the bacteria’s activities. The PCR test can come back positive or negative depending on the activity level of the infection.
http://www.tiredoflyme.com/4-reasons-a-lyme-test-will-come-back-negative-even-if-a-person-truly-has-lyme-disease.html

I'm not saying that PCR is a great test for detection of Borreliosis. It is a reliable test in the right hands but it has limitations.

I'll quote myself here

The main problem seems to be the low levels of bacteria in biological samples, the presence inhibitors and potential loss of bacteria from sample handling. These can all result in a potentially high number of false negatives.

What I objected to in the article you cited was the statement that PCR was not reliable because of problems with primers. This is not one of the limitations.

That statement is so wrong that it casts doubt on the credibility of the person making the claim rather than on PCR.

 

[duncan]

@alicec , do you know if you can broadly jerry-rig a PCR for the entire Borrelia genus?

 

[alicec]

do you know if you can broadly jerry-rig a PCR for the entire Borrelia genus?

I'm sorry I don't understand the question.

It should be possibly to find primers to amplify DNA from the entire genus (though I don't have any specific knowledge of this) but what does this have to do with jerry-rigging?

 

[Justin30]

So if all these Lyme tests prove to be poor and chronic Lyme disease is proven to not me real then likely you can add a massive cohort to the ME/CFS Group!

Another subtype...would that not be scary, are numbers would grow substantially.

I am not saying it isnt reel cause I believe it to be. But I think. Lyme is just another way to cripple someone and cause ME/CFS. I believe it to be in the co-infection category in disruption of the inate immune system. Im not a Dr though.

Similar to viruses in ME and messing up the whole immune system....

...the governments, NIH, health authorities, etc. Soon will be quivering in there pants as so many have come down with these vague illnesses that they have known about for years. It is starting to catch up to them they know it, I know it, the researchers and Drs know it....the suffering will end.

CFS/FM/LYME/POTS are at epedemic type levels....will multiple co-infections attach included.

 

[duncan]

@alicec, sorry, poor wording choice on my part.

One of the many problems with ANY Lyme testing is that the testing is calibrated for a single strain of a single species (in the US. In Europe it's calibrated to cover three species). So, it is argued that if you've the wrong species or a different strain than the test's, i.e., B31 or G39/40, the test may generate false results.

A way around this is to first employ a metric which determines if you've any Borrelia infection. Any. If yes, then parse down. If those more specific tests come up empty, clinicians or researchers - thanks to the genus-wide positive - know the patient is still infected; they may be simply missing the wrong strain.

Is there a way to target a PCR for the entire Borrelia genus?

That aside, I'm still not sold on the reliability of the PCR, although on paper I like the test. Neither, btw, is the IDSA, evidently. Some of its members will waste no time in denying a positive Bb PCR by claiming the positive is likely just due to debri.

 

[Dufresne]

@matsli

I'm wondering how prevalent these squiggly things are. Have you looked at the blood of many healthy individuals? As you pointed out, the three 'healthy' people in that study also had them.

Also, does anyone have any idea what percentage of sick and healthy people test positive by Elispot?

 

[Antares in NYC]

The lack of reliable tests for all things Lyme is one of the most puzzling and frustrating elements of this condition. The approved two-tier standard misses up to 50% of positives. Then the interpretation of a positive WB is at times so absurd as to require a ouija board to get your results, with many odd combinations where a positive result is often interpreted as a false positive. PCR in the US is extremely specific to BB Dna, missing any infection of other strains like garinii or afzelii. And we keep reading in the news the discovery of new strains, and even chimeras, hybrids between Borrelia types.

And then there are the seemingly astounding contradictions in the message from the authorities on Lyme, where we are told it's the fastest growing epidemic in North America, claiming 300,000 new cases per year (official CDC numbers) and told that about 20% of those affected never recover and suffer major damage.... But at the same time we are told it's not a serious infection and can be solved with two single weeks of antibiotics. The same researchers tell us of the dire consequences of neurological Lyme in one research paper, but then tell us that it only affects your knees in the next. The very same researchers that publish several papers on antibiotic persistent Borrelia then turn around and tell us that any lingering issues are "the aches and pains of daily living." They sure have brass ones.

I'll tell you this: if we had such shoddy, unreliable and contradictory tests for other serious infectious diseases like, say HIV, there would be riots in the streets. I don't doubt it for a second.

 

[matsli]

@matsli

I'm wondering how prevalent these squiggly things are. Have you looked at the blood of many healthy individuals? As you pointed out, the three 'healthy' people in that study also had them.

Also, does anyone have any idea what percentage of sick and healthy people test positive by Elispot?

@Dufresne


I have not made the study, I only initiated it. It is a biomedical scientist who made all work. We could not afford more than 5 healthy controls, because it is self-financed by the patients. All of the five showed no spirochetes.
https://newsaboutdisease.wordpress....onically-ill-have-spirochetes-in-their-blood/

In this study, the iSpot Lyme (Elispot in Europe) assay had a sensitivity of 84% and a specificity of 94%. Thats´s pretty good.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3972671/

 

[alicec]

Is there a way to target a PCR for the entire Borrelia genus?

Absolutely. In a quick trawl of the literature, many of the PCR assays targeted B.burgdorferi senso lato. Here is an example.

In this study they use nested PCR (amplification with one set of primers followed by amplification of this amplicon with another set of primers) to greatly increase the sensitivity of the assay. This gain in sensitivity is accompanied by an increase in likelihood of false positives so needs to be done very carefully.

Here is another example using a different target for amplification. Again nested PCR.

Other assays targeted other Borrelia species - eg B. lonestari.

It's possible to assay whatever you want depending on primer design.

It seems clear that PCR is a very useful tool for research. It has a place in diagnosis also and it seems there are ways of improving sensitivity to make it even more valuable. This however increases the technical demands.

 

[alicec]

I am not saying it isnt reel cause I believe it to be. But I think. Lyme is just another way to cripple someone and cause ME/CFS. I believe it to be in the co-infection category in disruption of the inate immune system.

I'm inclined to agree with you. Fits my experience anyhow.

 

[duncan]

@alicec - you may be right, but I'm concerned you use as examples Bbsl and lonestari. I'm not up on the science behind PCR, but was/is the logic of the primer based on three or four proteins, like OspA and p66? I cannot help but wonder knowing what we know now about shifting outer surface proteins that this might be logically problematic. But as I said, the science is admittedly beyond me.

Regardless, I mean testing at the genus level. That would include ALL Borrelia, including miyamotoi and close to 50 other Borrelia species. A one-test deal?

Now, if the answer is still yes, I will throw this out simply because it needs to be asked: Why isn't it being done now? (I realize you probably don't know, @alicec ). What would it take for say, the NIH, or Mayo, or any PCR vendor to bring such a test to market?

I would suggest if it isn't technology, then it's politics and market forces.

I just realized I am drifting from the thread's subject, so my bad.

 

[alicec]

but was/is the logic of the primer based on three or four proteins, like OspA and p66

The logic is simply finding an area of sequence which gives the specificity required and which is technically suited to the DNA binding requirements of the assay.

Those are just two targets which have been tried. Another is the 16S rRNA gene. There is an enormous amount of info about this gene from bacteria and many other organisms - it is the target for defining microbiome constituents so is a very robust.

Regardless, I mean testing at the genus level

I was just giving examples which can be quickly picked up from looking at the literature. Other species such as B. miyamotoi have been studied. Testing at the genus level would be possible also though I can't tell you anything about the technical difficulties/limitations of the approach.

As for why it isn't being done now, well perhaps because no-one think there is much advantage to it.

I'm not sure why you think such a test in itself would be the answer to a lot of problems. If widely applied it might help to define the prevalence of Borrelia infection in general but this would still need to be broken down into its constituent parts to understand the relationship of infectious agent and disease outcomes.

 

[duncan]

The answer to your question is: If your search is too focused, you may inadvertently (or not) exclude other answers.

An example would be if you go to the NIH and test for Lyme, you will only be tested for Bb sensu stricto. If I have been infected with garinii or afzelii, and have not been to Europe, the NIH will likely not check for those primarily-European species, and I will test negative. I will not receive treatment.

This is both a dogmatic and political problem - one that is played out over and over in clinical situations that are likely dealing with strain level issues, in addition to species - that might be circumvented with a first-line genus test.

If someone tests positive on a genus level, a clinician simply turning one's back on a patient's complaints because of a flawed conventional Lyme result becomes less of a viable option. The clinician will be forced to look more closely for a Borrelial cause, even though dogma dictates it's not supposed to be there.

Case in point: B miyamotoi. How many patients are infected throughout the US (and Europe, for that matter) that would not have been had a genus-level test been available these past five or so years?