Rebuttal published to CDC vs Advanced in J Clin Microbiology

Topics with information and discussion about published studies related to Lyme disease and other tick-borne diseases.
Pandora
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Re: Rebuttal published to CDC vs Advanced in J Clin Microbio

Post by Pandora » Wed 3 Dec 2014 8:35

I would not say contamination per say - as I would Expression and Methods used.

http://www.biomedcentral.com/1471-2180/11/6
Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus) microplus through tag-encoded pyrosequencing

While TX. can find upwards of 120 Bacterial genera -- China can find at least 370.

http://www.ncbi.nlm.nih.gov/pubmed/25150725

So something is either wrong in the methodology, the morphology, and or expressions.

How hard can some spirochetes be to culture? NO ONE has yet to culture Syphilis outside of a living host....Or have they?

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LHCTom
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Re: Rebuttal published to CDC vs Advanced in J Clin Microbio

Post by LHCTom » Wed 3 Dec 2014 22:51

The phone number to the Eurofins lab in Alabama is (256) 704-8200.

Tell them that you think the contamination in Sapi's culture took place at their lab.Please let us know what they have to say about that.
I'll call them when your IQ tops 100.

Well I for one don't trust what Sapi grew in those cultures.Of course,if someone who we all can trust does it,then we will know for sure if Sapi's culture works or not.
You probably don't trust your mother. I agree it needs independent unbiased validation. The CDC's poison paper didn't help.

Split the treated mice into 2 groups.One group gets cultured using standard BSK medium, the other group using the Sapi culture method.

This should not cost that much to do.I don't know why Sapi has not done this already,oh,oh, well maybe I do.
I know you have a limited imagination and struggle with concepts that are not black and white. Humans and mice are different. People are ill and can actually talk with us. So maybe we should just jump to culturing people. Oh that's right we already are doing that.

I dare one of the big brained scientists to just validate the Sapi culture. If it works great we helped many people. If not, maybe the big brains could make an effort at understanding the reasons Lyme and Syphilis can grow in vivo and not in BSK. It doesn't take a rocket scientist, although your IQ must top 100 to understand if something can grow in vivo, and you can mimic in-vivo properly, it will grow in vitro.I know this is tough to understand - If something grows and lives inside a living organism and you can exactly mimic those conditions, it will grow in vitro.

I would start with a transcriptome analysis since that's the most obvious possibility. It changes dramatically when it moves from the tick to the mouse and again back to the tick and again when to another different host like a human.

LCHTom can correct me if I am wrong,but what I hear him saying is the spirochetes transcriptome has changed at some stage of an infection and its needs to grow have changed.
Brilliant. No, that's one obvious possibility that has never been seriously investigated.
That's why Barthold and Bochenstedt can not grow viable spirochetes after abx treatment in standard BSK,but you should be able to grow them using the Sapi culture.Well, lets put that to the test.
Again, lets move on too people. Mice and humans have some useful similarities but they are far from identical. Its a lot cheaper and easier to get permission and risk liability testing ticks and mice than people.

Human IRBs are very different from animals and carry far less cost and risk.
Seventeen federal agencies have regulations governing the conduct of research involving human subjects. Examples of agencies with human subject requirements include the Department of Health and Human Services (DHHS), the Food and Drug Administration (FDA), the National Science Foundation, and the Departments of Defense, Education, Justice, and Veterans Affairs.

http://www.ncbi.nlm.nih.gov/pubmed/25425211
Much of Bochenstedt's,Wormser's,and the IDSA's Lyme opinions and guidelines are built around these animal models.Here is a chance to discredit all of that
That's one of the problems. You can do things to animals you can't do to humans. So its useful to take it as far as one can. But basing human treatment on animal models is stupid. Its a start but hardly adequate.

If you think that there is that much of a difference in the way Bb infects humans and animals,then why are all of these researchers wasting there time studying them?Do you think Dr Bochenstedt and Barthold are stupid for doing so?
Ok I'll go real slooooooowwwww. Animal models are useful and can provide useful information. You can do things to animals you can't do to humans like intentionally infect them. But what you learn must be taken in context. The transcriptome in a tick and mouse and human are all different. So you cannot assume what happens in a mouse is the same as in a human. I know its complex and difficult to imagine, but take a shot at it. It won't hurt and a little compassion might actually feel good.

Science is a process that moves forward. Yes forward. As technologies, understanding and techniques improve, what was believed true in the past regularly is found wrong. Think about that for just a moment.

BSK was developed and modified in the 80's and 90's and then stopped being improved. But microbiology and things like transcriptome undersatnding and analysis has been developed into being useful AFTER the BSK based culturing stopped evolving. Maybe just maybe science is following its normal course of moving forward...


Henry wrote
It is a well known fact that animal models of borreliosis in no way approximate the events that occur during Lyme disease in humans
Black and White thinking. There are similarities and differences. Its the people who can't think about the context and differences that are the problem.

Think about it.

You are a researcher and you have tradeoffs to make in trying to understand human disease.

Humans are the best since its the target and humans can communicate in subtle but important ways.

But you cannot experiment on humans due to ethical issues and the risk to the humans.

So depending on what you are studying and the limitations you are willing to accept, an animal is chosen.

A primate most closely mimics a human but have long lifetimes and cannot comunicate subtle information.

Mice are have short lifetimes and have biology that is closer to a human than a fish or birds.

But mice are natural Borrelia carriers which means the impact on their lives has worked and doesn't truncate their ability to survive normally and they support the spirochete life cycle along with ticks quite nicely. So mice have a major problem.

But mice have different immune systems and biology and put pressures on the spirochetes that only force their transcriptome to adjust to the necessary degree. The Transcriptome is the set of all RNA molecules, including mRNA, rRNA, tRNA, and other non-coding RNA transcribed that control how the DNA template is being implemented at any one time based on pressures and demands. These pressures inside a mouse are quite different from a human. The transcriptome is generally how the transcription of DNA is ongoing as needed or forced for an organsim to try and optimize its environment.

So if you could take a snapshot of all the transcription activity in a mouse at some phase of its infection it would be that which is necessary within the DNA template limitations at that moment. Everything in the mouse or host internal environmnet adjusts this transcription. It changes in an attempt to adapt to its environmental pressures and conditions.

Those are very different between a human and mouse or tick

A spirochete is able to live inside a mouse, primate or any host environment pressures and stresses by adjusting its transcription of genes that are helpful in symbiosis with the environment.

Its often discussed how the surface antigen upregulate and downregulate as they progress in a human infection. Thats the transcriptome adjusting. It doesn't only change surface antigen protein synthesis but everything necessary for the spirochete to survive. It must deal with a plathora of differences from an evolving immune system, an antibiotic onslaught, changes in temperatures, PH, toxins, and all the nutrients available to support life. A spirochete living in a tick, a mouse and human all vary wildly and vary over time and pressures.

So its absurd to assume that a culture medium condusive to survival and replication when a spirochete is removed from a tick or a mouse is the same as from a human after surviving years of this environment. If the culture media doesn't adequately mimic the environment from which it was taken, it most likley won't thrive.

So if you want to culture a human with a BSK media developed first for tick environments and then adapted for other hosts, it probably won't allow a spirochete to thrive after its adapted to a human host for a long time.

That's probably why researchers have been successful when culturing EM rashes because very little pressure has been applied and the spirochete is still similar to when it was in a tick as its only been in the fairly safe skin and for a short time. But as the spirochete moves to dissemination, the BSK media has generally failed. Why? There are either no spirochetes left, one hypothesis, or the spirochets have now been forced to adapt to the hostile human environmemt and the BSK and conditions no longer provides the environmemt to thrive.

So rsearchers have a few options. They can do the very difficult work of extracting disseminated spirochetes ( a catch 22) and analyze the transcriptome ( a very new evolving understanding) or they can get creative and guess as to the possibilities and resort to educated trial and error or experimental science like the fathers of science.

That's what Sapi did and it appears she may have had luck. But even if she didn't, if a spirochete can live in a human disseminated, than mimicing that environment will be invisible to the spirochete. It just may be difficult. But if a spirochete can live in one environment, if you can exactly duplicate it, how does it know its not in a human and it will grow. The trick is finding the formula its adapted to.

That's not deep science, its common sense.

So mice only go so far. Primates are better but are not identical and cannnot communicate to help in the process.

This relatively new science and I suspect the old timers are stuck in their ways. Maybe they need to get back to thinking like the father of Nuclear Physics Ernest Rutherford and get back to experimentation. The double slit experiment taught us more about the bizzare nature of reality than almost any other experiment.

Think about physics and astronomy. Much of what was belived truth has been overturned in the last 10 years. That true of all sciences. Its how it works. An understanding or theory or hypothesis is only as good as the evidence that underlies it. But as technology and tools and understanding and IMAGINATION evolve in microbiology and biology, that which was believed correct 10 years ago is constantly overturned. That's how science slowly and methodically moves toward current best truth but will never get reach the limit.

Physics was stuck on Newtonian physics for over 200 years. Then Einstein came along and over 30 years everything changed. Even Einstein got stuck believeing God didn't throw dice and couldn't accept Quantum Mechanics which now underlies many real technologies from MRIs to Solar Panels. Until you get it, you might as well go to church and put your head in the sand.

Nnecker and Henry

hv808ct is a beast of a different color. He is one of the old guard in deep fear driven concealment and clearly has deep personal reasons to behave the way he does. Its so sad. I guess compassion is not something everyone is born with. Go see the movie "The Theory of Everything". Its a great lesson in how humans can evolve beliefs if driven by compassion and hardship. If you leave the movie dry eyed, you are not alive.
The greater the ignorance, the greater the dogmatism.

Attributed to William Osler, 1902

Henry
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Re: Rebuttal published to CDC vs Advanced in J Clin Microbio

Post by Henry » Thu 4 Dec 2014 14:20

LHCTom : You said the following : " BSK was developed and modified in the 80's and 90's and then stopped being improved. " However, if you bothered to read the paper by Barbour et al., a reference cited in the paper by Sapi et al., you will find that Barbour et al. were able to culture from SINGLE CELLS of Borrelia on BSK . I'd say that's pretty darn good -- hard to improve on that really. Aside from the molecular issues and problems related to contamination, Sapi et al. did not rigorously compare their results -- head to head using clinical isolates of Borrelia-- to those obtained using classical BSK medium to enable one to determine if their "new" method is really more than just a modest advance at best. And, that's giving Sapi et al. far more credit than they deserve.

If one is unable to culture Borrelia from patients who believe that they have Lyme disease, it just might be that they don't have Lyme disease in the first place, or the culturing was done at the wrong time. In either case, it does not mean that BSK medium is "no good". It is good enough to do the job IF Borrelia are present.

duncan
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Re: Rebuttal published to CDC vs Advanced in J Clin Microbio

Post by duncan » Thu 4 Dec 2014 14:40

Henry: "It is good enough to do the job IF Borrelia are present."

Well, therein rests the problem, else why would we need all those annoying antibody tests? The fact is Borrelia have this nasty tendency to go MIA in people who are 100% infected, rendering the old culture pretty much useless in a majority of cases once you leave the EM comfort zone. Which is why building a better culture mechanism that could secure reliable results outside of an EM consistently , from early stage through late stage disseminated, would be a good thing.

nnecker
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Re: Rebuttal published to CDC vs Advanced in J Clin Microbio

Post by nnecker » Thu 4 Dec 2014 18:43

LCHTom said
You probably don't trust your mother.
If my mother was Eva Sapi, your right I would not trust her.However she is not, and I did trust my mother very much when she was alive.But by looking at what a class act you are by talking about other peoples mothers, I can honestly say I would not trust your mother just by looking at what came out of her.

velvetmagnetta
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Re: Rebuttal published to CDC vs Advanced in J Clin Microbio

Post by velvetmagnetta » Thu 4 Dec 2014 21:07

Now, now, boys. Let's stick to the topic and leave each others' beautiful mothers out of it.

Henry, you said:
...Barbour et al. were able to culture from SINGLE CELLS of Borrelia on BSK . I'd say that's pretty darn good -- hard to improve on that really. Aside from the molecular issues and problems related to contamination, Sapi et al. did not rigorously compare their results -- head to head using clinical isolates of Borrelia-- to those obtained using classical BSK medium to enable one to determine if their "new" method is really more than just a modest advance at best....
Ah, but she did compare BSK alongside other combinations. See figure 1.

http://www.medsci.org/v10p0362.htm

Click on the picture to enlarge.

And remember, this is just one paper on one study on the improvement of culturing Borrelia. She did not, and was not supposed to, answer every question everybody has about the mysteries of Lyme disease!

Give her a break. You can't measure too many things in one experiment - otherwise your experiment won't mean much. The more specific it is, the more you can rely on its results.

Now is the time for Sapi or someone else to take this culture one step further and see of you can get any spirochetes from antibiotic-treated blood samples.

I predict we will not be able to grow any from treated blood. But let's find out for sure.

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LHCTom
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Re: Rebuttal published to CDC vs Advanced in J Clin Microbio

Post by LHCTom » Fri 5 Dec 2014 6:11

Henry said:
BSK was developed and modified in the 80's and 90's and then stopped being improved. " However, if you bothered to read the paper by Barbour et al., a reference cited in the paper by Sapi et al., you will find that Barbour et al. were able to culture from SINGLE CELLS of Borrelia on BSK . I'd say that's pretty darn good -- hard to improve on that really.
That means nothing. All living cells are based on DNA which is the template or genome which is essentially fixed. But how DNA is expressed is something that can adapt to external conditions in order to optimize the liklihood of survival. Thats very roughly what a transcriptome is. So the SINGLE CELL that was grown in BSK had a transcriptome or gene expression control state from its most recent environment. So if this one celled Borrelia was from a tick or purchased from a supplier, it had a transcriptome that had been adapted to its environment. So one Borrelia cell is NOT the same as another Borrelia cell.

Its very clear that the BSK developements in the 80' and 90's were successful at providing the environment sufficiently similar to that of a tick Borrelia transcriptome or mouse transcriptome or even the early stages of a human infection. But as environmental pressures are applied to this Borrelia cell, the control portion of the genome tries to adapt to survive in the new environment. Once the Borrelia cell leaves the EM rash and begins dissemination, it encounters a massive immune response, an antibiotic assault and as it moves into new homes in the human, that gene expression does its best within the limitations of the genome to adapt and survive. Even its morphology can change in an attempt to survive. You have probably heard of up-regulation and down-regulation of surface antigens, well that's just one samll example of an adapting transcriptome.

So just because the BSK brew Barbour et al and the gang cooked up grew the ONE CELL, of unknown origin, it doesn't mean that same brew or formula and conditions will grow another Borrelia cell that's been adjusting its transcriptome for years in a human dissemination because ITS DIFFERENT. The only way to know is extract a Borrelia cell from a human after years of environmemtal onslaught and try and grow that cell. But every Borrelia or bacteria of any kind that is found somewhere in the human body is probably different. Have you ever wondered why seriously pathogenic bacteria live in your gut or nasal or elsewhere in your body and rarely cause problems? Well one reason is they have adapted to the environment and part of that is how they have adatped their transcriptome. Its an ever changing state of gene expression control.

So your ONE CELL statement means nothing. I'm not saying its the guaranteed reason, BSK doesn't seem to grow disseminated Borrelia post treatment successfully, but its a plausible hypothesis that is quite reasonable that you MUST consider before declaring a particular culture design "perfect" "done" and there is no point in trying to match the culture to the new needs of of the bacteria. Most bacteria is not culturable. But we know it grows quite nicely in some environments. So what does that suggest. We are unable to duplicate the conditions required for that bacteria to grow in vitro because we don't understand that environment sufficiently to duplicate where its known to live quite nicely. Its not that bacteria are unculturable, its we don't know how to duplicate what it needs. There are other possiblities but when a scientist stops using imagination and creativity, progress generally stops or slows down. That's what is going on here in part.

Not Black and White - complicated shades of grey!
The greater the ignorance, the greater the dogmatism.

Attributed to William Osler, 1902

Henry
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Re: Rebuttal published to CDC vs Advanced in J Clin Microbio

Post by Henry » Fri 5 Dec 2014 13:46

LHCTom: Oh yes, you have quite a vivid imagination. However, it doesn't serve you well since -- because of your strong bias and ego-- you can't seem to see the forest because of all the trees. What Barbour and colleagues did was -- by a limiting dilution assay-- take a single Borrelia cell derived from an infected mouse, or a tick, or a human and cultivate it to large numbers in BSK medium. That is a rather simple and straightforward experiment, indicating that Borrelia derive from such environments are able to grow perfectly well -- from very small numbers-- in BSK. No need to resort to a rather long and irrelevant discourse on the transcriptome, unless you feel the need to convince others of how smart you (think you) are. Yawn, yawn....

duncan
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Re: Rebuttal published to CDC vs Advanced in J Clin Microbio

Post by duncan » Fri 5 Dec 2014 14:18

So the old culture is not a good tool for capturing Borrelia outside of an EM. It is not adequate due to limitations that include the small number of Borrelia in patients' blood as the disease disseminates. Also because the spirochete morphs, making it difficult to culture and reproduce in vitro. So, there is a need for a better tool for direct cultivation. Until such a tool is crafted, we must rely on antibody tests with questionable authority. Unless the Sapi method IS that better tool, and we are still waiting on validation of that product.

Henry, you yawn a lot. You may wish to have that looked at. Possible CFS case? If you'd like, I can recommend a good clinician.

Henry
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Re: Rebuttal published to CDC vs Advanced in J Clin Microbio

Post by Henry » Fri 5 Dec 2014 14:34

No need to bother making a recommendation, Duncan. It is due to boredom -- from hearing the same old unsubstantiated claims and speculations being made over and over again -- as though they were fact-- with no evidence to support them. Now, don't excuse them because of your medical problems.

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