Resurgence of Persisting Non-Cultivable B burgdorferi

Topics with information and discussion about published studies related to Lyme disease and other tick-borne diseases.
ChuckG
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Resurgence of Persisting Non-Cultivable B burgdorferi

Post by ChuckG » Fri 24 Jan 2014 1:11

http://www.plosone.org/article/info%3Ad ... ne.0086907

Resurgence of Persisting Non-Cultivable Borrelia burgdorferi following Antibiotic Treatment in Mice
Emir Hodzic, Denise Imai, Sunlian Feng, Stephen W. Barthold

Published: January 23, 2014DOI: 10.1371/journal.pone.0086907
Abstract

The agent of Lyme borreliosis, Borrelia burgdorferi, evades host immunity and establishes persistent infections in its varied mammalian hosts. This persistent biology may pose challenges to effective antibiotic treatment. Experimental studies in dogs, mice, and non-human primates have found persistence of B. burgdorferi DNA following treatment with a variety of antibiotics, but persisting spirochetes are non-cultivable. Persistence of B. burgdorferi DNA has been documented in humans following treatment, but the significance remains unknown. The present study utilized a ceftriaxone treatment regimen in the C3H mouse model that resulted in persistence of non-cultivable B. burgdorferi in order to determine their long-term fate, and to examine their effects on the host. Results confirmed previous studies, in which B. burgdorferi could not be cultured from tissues, but low copy numbers of B. burgdorferi flaB DNA were detectable in tissues at 2, 4 and 8 months after completion of treatment, and the rate of PCR-positive tissues appeared to progressively decline over time. However, there was resurgence of spirochete flaB DNA in multiple tissues at 12 months, with flaB DNA copy levels nearly equivalent to those found in saline-treated mice. Despite the continued non-cultivable state, RNA transcription of multiple B. burgdorferi genes was detected in host tissues, flaB DNA was acquired by xenodiagnostic ticks, and spirochetal forms could be visualized within ticks and mouse tissues by immunofluorescence and immunohistochemistry, respectively. A number of host cytokines were up- or down-regulated in tissues of both saline- and antibiotic-treated mice in the absence of histopathology, indicating host response to the presence of non-cultivable, despite the lack of inflammation in tissues.

Claudia
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Re: Resurgence of Persisting Non-Cultivable B burgdorferi

Post by Claudia » Fri 24 Jan 2014 22:23

Thanks for posting this, ChuckG.

Margherita
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Re: Resurgence of Persisting Non-Cultivable B burgdorferi

Post by Margherita » Sat 25 Jan 2014 1:13

but persisting spirochetes are non-cultivable
Why not?

ChuckG
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Re: Resurgence of Persisting Non-Cultivable B burgdorferi

Post by ChuckG » Sun 26 Jan 2014 1:51

http://relative-risk.blogspot.com/2014/ ... vable.html

Relative Risk's take on this paper.

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LHCTom
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Re: Resurgence of Persisting Non-Cultivable B burgdorferi

Post by LHCTom » Sun 26 Jan 2014 5:52

but persisting spirochetes are non-cultivable
Why not?
Most bacteria cannot be cultured. All Borrelia species are not equally cultivable. The syphillis spirochete has never been successfully cultivated. The ability to culture a bacteria or a Borrelia spirochete requires duplicating in-vitro a sufficiently non-hostile environment and medium that provides whatever the host provides. This has been accomplished in Borrelia burgdoferi but with its original set of wild genes. The spirochetes can completely lose plasmids containing genes required for culturing but are still able to perform sufficient transcription to replicate only now in-vivo. Bacteria and Borrelia spirochetes are also known to horizontally transfer plasmids. This means the spirochete could take on "new" genes altering their being cultivable. The pressures of a combined antibiotic treatment and the immune response could lead to the survival of spirochetes with "missing" or permanently down-regulated genes. Maybe they have become truly un-cultivable or the requirements for culturing are significantly different such that existing culturing techniques are inadequate. So there are a number of possible changes at the genetic level that could alter "being cultivable ".

Among the most important observations in this study are the resurgence and continued ability to transcribe DNA into mRNA. The idea that the spirochetes are no longer cultivable is not terribly important. Its a bummer because prior to the ability to do mRNA PCR, there was no way to tell if the symptoms were caused by a living organism that is persisting or left over DNA junk that can survive a very long time without a culture proof its living. There are multiple mechanism that protect DNA from degradation and actually repair damaged DNA. So old DNA can hang around for a long time. On the other hand, mRNA degrades fairly quickly in-vivo since there are multiple mechanisms to make this happen intentionally. mRNA is transcribed, used for producing a protein or to control another gene expression and then destroyed. So the continued and increasing mRNA for multiple genes is similar evidence that the organism is living as being able to culture it.

To my knowledge, there has yet to be a serious attempt to use mRNA PCR to look for living Borrelia in humans but its become a widely used technique that when combined with nested DNA PCR, should be very sensitive. mRNA is reverse transcribed into cDNA and then the nested PCR can be used to detect the cDNA in a manner identical to the well known PCR. Nested PCR uses outer and inner primers to greatly increase sensitivity and is commonly used now when testing humans and ticks for Borrelia and has been shown to be able to detect a few or even a single spirochete piece of DNA while standard PCR might need hundreds or thousands.

Being un-cultivable has been seen in many studies and there are many viable possible reasons at the genetic change level for this as a result of treatment/immune pressures. Being un-cultivable does not mean they cannot replicate and disturb the immune system causing inflammation which causes symptoms. Its hard to ask the mice if they feel neurological symptoms. They typically don't answer when asked. Humans can describe effects of fairly minor brain inflammation that can't be observed in dissected mice. The increased cytokine detection when compared with infected mice supports this also. This probably means the spirochetes are no longer able to infect another host easily but is doesn't mean these down-regulated un-cultivable spirochetes cannot continue to cause human CNS inflammation and the symptoms seen in chronic Lyme. The resurgence after 12 months means they must have come out of down-regulation or hiding and begun replicating and then detecting Borrelia mRNA for multiple genes also supports a resurgent persistent infection. The spirochetes are just a bit genetically down regulated and no longer wild. It requires a living and surviving organism for the transcription from DNA to mRNA for multiple genes to be occurring. It isn't necessarily genetically complete like the original wild spirochete.

So don't get hung up on the "un-cultivable" as it does not mean the spirochetes are dead and its not a persisting infection. This study is solid evidence that persistence in mice is real after antibiotic treatment. Its the resurgence and detection of mRNA that are new important evidence. This may very well be what is happening in humans. This also suggest mRNA is an important technique that should be pursued when looking for persistence in humans because it won't be fooled if the spirochetes are un-cultivable as seen here and before.
The greater the ignorance, the greater the dogmatism.

Attributed to William Osler, 1902

Margherita
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Re: Resurgence of Persisting Non-Cultivable B burgdorferi

Post by Margherita » Sun 26 Jan 2014 16:32

Dear LHCTom,

One of the reasons I was wondering why persisting Borrelia shouldn't be cultivalbe was the Embers monkey study where PCR detection (with culturing I suppose) was one of the methods used to discover the persiting infection after treatment.

Thank you so much for your detailed explanation. Things are a lot clearer now. I might be wrong, but wouldn't it have been better to write the following:
but not all persisting spirochetes are cultivable
??


At this point I'm still wondering why instead of investing all money and efforts in a new Lyme vaccine, development of a new reliable PCR test for patients, if possible with both, RNA and DNA detection, doesn't seem to have any priority :roll:

Kind regards and have a nice Sunday
Margherita

edbo
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Re: Resurgence of Persisting Non-Cultivable B burgdorferi

Post by edbo » Sun 26 Jan 2014 17:52

LHCTom, thank you so much for your explanations and for breaking things down into digestible pieces of information. For some of us laypersons this is always very helpful!

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LHCTom
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Re: Resurgence of Persisting Non-Cultivable B burgdorferi

Post by LHCTom » Sun 26 Jan 2014 20:28

There is a pathologist named Sin Hang Lee at MIlford Hospital in Conn. that is offering a combination nested PCR combined with sequencing that should be far more sensitive than standard PCR. The nested PCR can be up to a thousand fold more sensitive than standard 2 primer PCR if done carefully. Its the first time I'm aware of that nested PCR and sequencing is being offered for the detection and identification ( of strain and species) to mainstream doctors.

http://www.dnalymetest.com/lymediseasediagnostics.html

This is more interesting than usual because Sin Hang Lee has published papers giving his lab more credibility and a level of validation. The technique itself does not need validation since its common used and widely accepted. The question would be in whether a particular lab was sloppy. Nested DNA PCR must be done very carefully and the primer pairs chosen very carefully. The choice of the 16S ribosomal RNA gene is also a common approach and by Next-Gen sequencing the PCR results and then using the NCBI BLAST, it can be shown its really Borrelia DNA and its strain and species identified. If the sensitivity is as good as shown in the paper, we might finally have a PCR test for Lyme that has good sensitivity but also shows real human infection strains in the general population rather than a small human study.

Its also well known that nested PCR vastly outperforms standard PCR in detecting small amounts of DNA. So there is no magic here. They are simply using the best tools commonly used and validated. So this might be the "real deal" - University level tools available to regular doctors. They are almost certainly capable of doing the mRNA PCR so maybe that's next as was used in the Barthold mice study. Since the mRNA PCR requires the removal of all normal DNA followed by reverse transcription of the mRNA back into cDNA, it may not have the sensitivity to pick up small amounts of mRNA. Every cell has a complete set of DNA genes and plasmids and they are very stable. mRNA is only transcribed when a gene is expressed and then its quickly degraded intentionally after its used to build a protein or control the expression of another gene. So there is a lower volume to find and the choice of which mRNA is important. But when mRNA is reverse transcribed, its converted into cDNA which is just the complementary set of base pairs. So it then becomes identical to normal DNA.

http://ajcp.ascpjournals.org/content/133/4/569.abstract

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2984391/

Just like any lab test, if Borrelia contamination gets in the sample being analyzed, it will happily show the strain and species but be wrong. The good news is if they find strains and species that properly match what has been found in the past, its good evidence for the lack of contamination. For example, here in CA we have strains of Bb that are quite different from those more commonly found elsewhere. There has been enough tick Bb strain testing within each endemic state for there to be fairly good statistical idea of what strains are common. So if the labs detection of strains even roughly matches the known statistics of strain distribution, its strong evidence its not contamination. If they contaminate their samples somehow, the test results will cluster around the contamination source. Most Next-Gen Sequencing errors rates can be as high as the 1/2% range ( depending on how its done) so for every 200 base pairs of DNA, it would be possible to find 1 base pair error - very very roughly. I'm assuming they can't afford doing the most accurate techniques for a few hundred $ lab blood test. But even a single base pair error won't typically interfere with the strain ID.

http://www.dnalymetest.com/dnasequencetesting.html

This offering is also important because nested PCR if done correctly and without contamination is far more accurate for diagnosing Lyme disease. It will finally be able to provide data on which strains and species like B miyamotoi are really infecting humans as is being done by Kerry Clark. The main difference between this lab and Kerry Clark's search with nested PCR was he looked for the flaB gene. The nested PCR testing approach used by Kerry Clark and that described by Sin Hang Lee this are similar. Kerry Clark's results have been important because he has shown that Borrelia burgdorferi is not the only species found in the US along with the recent finding of B miyamotoi in humans. The California Health Department's Dr Padget has found about 1/2 the ticks tested positive for Bb also were infected with B miyamotoi. But to date, there have been no California studies testing humans for B miyamotoi using nested PCR and the antibody response to B miyamotoi and other distant strains or species is missed on the antibody tests.

http://www.medsci.org/v10p0915.htm

http://www.cdph.ca.gov/services/boards/ ... -15-13.pdf


Everybody here is aware of the numerous problems with the antibody based tests and the low sensitivity of standard PCR. I'm guessing this approach will become the replacement for the 2 tiered antibody test. Unfortunately in late disseminated lyme disease, the number of spirochetes in the blood could be very low to zero at times. But say someone was infected 10 years ago and treated 7 years ago. Even though the Borrelia DNA can persist in humans for a long time, its unlikely it will persist long in blood. Its probably going to persist in tissues or immune privileged places and rarely escape into the blood unless its replicating. So if someone treated 7 years ago is found positive for Lyme by PCR, its not proof of replication persistence but suspicious evidence. If one finds Borrelia DNA commonly in people treated many years ago and its suspected of being non-cultivable, it will become obvious to microbiologists to try mRNA PCR of multiple genes.

But its ALWAYS critical to avoid contamination so this should be interesting. It should be easier to avoid contamination with PCR over a culture given all else equal because the PCR test is done overnight while a culture needs to sit nearby other cultures for as long as a month. So the PCR test contamination exposure should be easier since it arrives and is tested versus the culture hanging around hundreds of high volume growing spirochetes from other cultures and in a commonly made culture medium. There is just more opportunity in the culturing environment but it obviously can and has been done without contamination successfully. I bet this is just the tip of the iceberg as other labs decide to follow the lead of Sin Hang Lee. It could be the beginning of the end of the testing nightmare.
The greater the ignorance, the greater the dogmatism.

Attributed to William Osler, 1902

lou
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Re: Resurgence of Persisting Non-Cultivable B burgdorferi

Post by lou » Mon 27 Jan 2014 0:25

It doesn't matter whether this is the best test or not, if the CDC is enforcing the two tier system and claiming that all PCR type testing is too subject to contamination to be trusted. We aren't just up against difficulties of finding Bb, but even worse, against an ignorant bureaucracy that does not want a good test.

And if spirochetes are found in tissues rather than blood in chronic and late stages, then a PCR test of blood, nested mRNA, may still come up false negative.

Anyone want to bet on how long it will take the CDC or CT health dept to come down on that new PCR test and the Milford Hospital?

Margherita
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Re: Resurgence of Persisting Non-Cultivable B burgdorferi

Post by Margherita » Mon 27 Jan 2014 0:58

Thank you once again LHCTom.
I'll read it over a few more times and I hope you do not mind if I'll come back with some more questions.

Lou wrote:
It doesn't matter whether this is the best test or not, if the CDC is enforcing the two tier system and claiming that all PCR type testing is too subject to contamination to be trusted
The contamination matter in PCR testing always seems to be a much bigger issue when Lyme disease is involved!

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