Improved Borrelia burgdorferi Blood culture Method

Medical topics with questions, information and discussion related to Lyme disease and other tick-borne diseases.
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Re: Improved Borrelia burgdorferi Blood culture Method

Post by RitaA » Tue 3 Sep 2013 5:55

LHCTom wrote:I for one, just want the truth.
Me too, and I want to join those who have already thanked you for helping the rest of us better understand the nitty gritty details involved.

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Re: Improved Borrelia burgdorferi Blood culture Method

Post by inmacdonald » Thu 5 Sep 2013 15:51

PyrG Haplotype Analysis : Truth or Consequences for the CDC

When DNA nucleotide sequencing Prints "Y" instead of "A" C" "G" T"
the identity of the "Y" nucleotide postition is UNDETERMINED.

We do Not DISCARD "y" mismatches as "irrelevant" or "non-contibutory"

BLASTn is an analytical tool which is politically neutral.

A "y" nucleotide is an Undetermined Nucleotide.

"Y" nucleotide mismaches in BLASTn searches will generate a Mismatch message, pure and simple.

For the CDC to report 100% identity when BLASTn supercomputer reports 99% match or less
is evidence that the CDC employed Politics rather than :"Hard Science"; in the writing of their paper

BLASTn searches of other geographies demonstrate borrelia strains which are temporally and geographicaly
DIVERSE, but which share Percentages of nucleotide identiies.
Are all supercomputer searched using BLASTn data for other Borrelia GenBank
deposits showing Identical Nucleotide sequences :
If 100% identities are discovered by BLASTN among a series of Borrelia Deposits; What Proper Conclusions accrue?
If a 92% identity for a GenBank deposit is discovered by BLASTN : What Proper Conclusions accrue?
Are all 100% identity group members "Contaminated":...virtual "victims" of Cross Contamination by the reporting Laboratories? I think that this is absurd.
Are all 92% identity group members ....Prima FAcie evidence of Simultaneous Contamination by Two other GenBank
borrelia Deposits? --I think that this is absurd.

Spontaneous mutations in borrelia are well known to all.
"contamination" does not scientifically find the Root cause of ANY Spontaneous borrelia Nucleotide
diversity with respect to known and completely sequenced Reference borrelia named strains.

Is there a previous peer reviewed publication on the topic of the use of pyrG HAPLOTYPE alignments for
a series of borrelia of any named type or for any microbe of any named type.
In short: is pyrG DNA alignment data from BLASTn either
1.Highly useful in detecting issues of alleged specimen Contamination?
2. Not all at useful in analyzing issues of alleged specimen Contamination?
3. An accepted "Benchmark " tool among Career Molecular biologists for Any type of ratification of the Validity of
any GenBank Depost.
4.Are pyrG Haplotypes equivalent to a forensic tool for discovery of "Counterfeit" from Genuine
Genbank Submissions.
5. Is there a Published peer reviewed precedent for Simultaneous "Two strain contamination of a single borrelia
specimen " with subsequent DNA sequencing for Haplotype pyrG nucleotide sequences?

In the Absence of Precedents for issues 1-5
No sound scientific Conclusions can be drawn by the CDC based solely on Haplotype borrelia pyrG data.

Alan B. MacDonald MD
Sept 5,2013

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Re: Improved Borrelia burgdorferi Blood culture Method

Post by edbo » Fri 10 Jan 2014 17:46

It has been fairly quiet in here... Does anybody have any new information concerning the blood culture method?

-Are there any results of the other independent validations in labs such as Maine Medical Center, Portland and UCLA, California?
-Is there an official response from Sapi et al. concerning the issues of alleged lab contamination described by the CDC?

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Re: Improved Borrelia burgdorferi Blood culture Method

Post by LHCTom » Mon 13 Jan 2014 20:30

Its oddly quiet. Advanced Laboratory did take down the paper and most of the Borrelia culture claims. I suspect they must be concerned that the FDA will do to them what they did to 23andme. If ALS had a scientifically solid response, they would have made it public rather than eliminate their culture claims. A major concern is whether the FDA is considering shutting ALS down rather than allowing them to work out the kinks. There are quite a few people who have been diagnosed with Lyme based on the culture without any other serological evidence. There are people with no Western Blots bands at IGenex who are now being treated. The FDA will frown on this. A Lyme culture would be very valuable but ALS messed up. Its a bummer but true. I'm concerned this will put a stop to Lyme culture advancement due to some laboratory contamination. We know the Lyme culture can work and we know Borrelia is the cause of Lyme disease. So this is not like the CFS XMRV debacle where XMRV has nothing to do with CFS. Borrelia does cause Lyme.

There is little doubt the first attempt at validation was flawed. You don't need to do a DNA pyrG gene comparison with the test strains to see there was serious problem. You don't even need the Johnson analysis. The 72 Lyme patients were recruited from 15 US states including CA, AR, CT, NH, NJ, NY, MA, MD, MN, OR, PA, TN, UT, VT. So they were drawn from a wide geographical range suggesting fairly diverse strains. Of the positive 51 samples, the ALS paper phylogenetic tree shows 27 of the 51 were a Borrelia garinii strain. This is not contended. This would be a major Borrelia scientific study finding over 50% B gariniii in humans given B garinii has never been found in 1 US patient ( infected in the US), or a US tick of many thousands tested or any host reservoir. Just finding over 50% B garinii is implausible. It should have been a big red flag for ALS and they should have stopped and investigated.

Of the 27 B gariniii, 21 had identical pyrG genes meaning they are likely closely related since a search of the NCBI database shows very few Borrelia garinii strains have identical pyrG genes. Out of dozens of B garinii strains collected over years, only the PBi strain matches the 21 identical pyrG genes. The 21 identical Borrelia garinii are a match to the Asian Fuji P1 strain based on an NCBI lookup. For example: another Asian Fuji P2 strain pyrG gene is 97% or 587/603 relative to the Fuji P2 strain - 16 different nucleotides out of 603. Finding 21/51 identical B gariniii pyrG genes is unimaginable.

Ok, so the original study is flawed and ALS has taken down all their claims from their website. None of this means a culture wouldn't be incredibly valuable tool for clinicians. Even Wormser achieved nearly 50% serum sensitivity ( during early EM infection) so its not inconceivable that with more blood and better technique, a useful sensitivity should be possible. Hopefully the next news we hear isn't the FDA coming down on ALS but its likely. I suspect ALS is in communication with the FDA and asking to hold off till the other studies come in. Its unfortunate that this first ALS study had so many problems - at least half. I suspect the other studies are carefully looking at sources of contamination ( e.g. growth medium being moved between sites, etc...) . If you eliminate the obvious contamination of B garinii, the real sensitivity of the ALS culture technique is probably going to be much lower except in early disease while the spirochetes are in the blood. Once someone has been treated and the spirochetes go into hiding, their gene expression downshifts and the immune response comes up, finding spirochetes in blood will probably be difficult even if they persist in tissue and organs. That is my suspicion but a culture would still be valuable given the testing nightmare that exists with Lyme.

I would like to see a very sensitive mRNA PCR attempted after months to years because there is a good chance the spirochetes are no longer cultivable due to altered gene expression. Why mRNA? Pieces of DNA could float around inside someone for quite some time. Those pieces of DNA would be good evidence of a former infection but pieces of DNA can last a long time. The machinery required for transcription of DNA into RNA is a complex multi-step process requiring a living organism. RNA is not very rugged like DNA. The machinery protects DNA but actively degrades RNA once its used. So DNA can last a long time but RNA should degrade in days to weeks. Once the last transcription from DNA to RNA has occurred in a living organism, that RNA will degrade. So if someone has been treated and the IDSA claims they do not suffer from persistence and Borrelia RNA is found months or years later, there must be living Borrelia in some form. So if Borrelia becomes uncultureable in Vivo, a culture cannot work but there is no mechanism possible for Borrelia stopping all transcription - So RNA PCR may be the best strategy for a persistence search.
The greater the ignorance, the greater the dogmatism.

Attributed to William Osler, 1902

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Re: Improved Borrelia burgdorferi Blood culture Method

Post by inmacdonald » Sun 2 Feb 2014 1:35

Please Post the link to the manuscripts which demonstrate

different kinetics of DNA degradation in the extracellular compartment in a life form

as opposed to the Kinetics of degradation of mRNA in an Extracellular Compartment in a life form.

Alan B.MacDonald, MD FCAP,,FASP
Feb 1, 2013

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Re: Improved Borrelia burgdorferi Blood culture Method

Post by LHCTom » Mon 3 Feb 2014 2:05

Not sure of the purpose or intent of the question but the comparison between the degradation of mRNA versus DNA is not terribly relevant since one would be looking for an increase in mRNA indicating living organisms are actively transcribing. If an organism is living or re-surging as was seen in Barthold's recent paper, its the increase in mRNA that is most meaningful. If the organism is truly dead, the transcription of DNA into mRNA stops. From that time, the organisms DNA and mRNA would degrade at different rates in different niches in the body. So if one wants to prove a living organism is present or preferably dividing and increasing, the mRNA would increase. Should the organism be dead, it cannot transcribe mRNA so the mRNA and DNA would begin degrading at rates depending on location and many factors. But it can only increase if there is active transcription.

In reviewing papers attempting to use mRNA to detect ongoing infections, they routinely mention the half life of mRNA as being quite short such as:
mRNA is turned over rapidly in living bacterial cells, with most mRNA species having a half-life of only a few minutes (2, 7). Detection of mRNA might therefore be a good indicator of living cells or those only recently dead at the time of sampling. ... =reader#B7
2. Alifano P, Bruni C B, Carlomagno M S. Control of mRNA processing and decay in prokaryotes. Genetica. 1994;94:157–172. [PubMed]
7 Belasco J. mRNA degradation in prokaryotic cells: an overview. In: Belasco J, Brawerman G, editors; Belasco J, Brawerman G, editors. Control of messenger RNA stability. San Diego, Calif: Academic Press, Inc.; 1993. pp. 3–12.
Irrespective of this, the presence of a living organism should cause the presence of mRNA to remain constant to increasing, while dead organisms will lead to declining DNA and mRNA and most sources suggest mRNA declines quite quickly.

If Borrelia becomes non-cultivable, this is the only strategy has has a chance of showing persistence. his technique has been used for ongoing infection detection of other bacteria other than Borrelia. Barthold used the technique in mice recently with success.
Detection of mRNA by Reverse Transcription-PCR as an Indicator of Viability in Escherichia coli Cells ... =reader#B7
Detection of Periprosthetic Infections With Use of Ribosomal RNA-Based Polymerase Chain Reaction ... po=92.6471
The greater the ignorance, the greater the dogmatism.

Attributed to William Osler, 1902

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Re: Improved Borrelia burgdorferi Blood culture Method

Post by RobertF » Mon 3 Feb 2014 11:40

Lazarus found that DNA is cleared within hours
Clin Dev Immunol. 2012;2012:138069. doi: 10.1155/2012/138069. Epub 2011 Oct 26.
ELISA-based measurement of antibody responses and PCR-based detection profiles can distinguish between active infection and early clearance of Borrelia burgdorferi.
Lazarus JJ, McCarter AL, Neifer-Sadhwani K, Wooten RM.
Author information
Borrelia burgdorferi is a spirochetal bacterium that causes Lyme disease. These studies address whether current research methods using either ELISA to detect seroconversion to B. burgdorferi antigens or PCR quantification of bacterial DNA within tissues can accurately distinguish between a productive infection versus a B. burgdorferi exposure that is rapidly cleared by the innate responses. Mice receiving even minimal doses of live B. burgdorferi produced significantly more B. burgdorferi-specific IgM and IgG than groups receiving large inocula of heat-killed bacteria. Additionally, sera from mice injected with varied doses of killed B. burgdorferi recognized unique borrelial antigens compared to mice infected with live B. burgdorferi. Intradermal injection of killed B. burgdorferi resulted in rapid DNA clearance from skin, whereas DNA was consistently detected in skin inoculated with viable B. burgdorferi. These data indicate that both ELISA-based serological analyses and PCR-based methods of assessing B. burgdorferi infection clearly distinguish between an established infection with live bacteria and exposure to large numbers of bacteria that are promptly cleared by the innate responses.

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Re: Improved Borrelia burgdorferi Blood culture Method

Post by inmacdonald » Mon 3 Feb 2014 16:05

Half Life Kinetics papers - DNA , RNa now posted.

The next question in this interrogation and reconciliation process is:

Extracellular DNA Degradation Kinetics:
Intracellular DNA ( foreign to host) - Is there a Kiinetics of DNA (Intracelluar type) Degradation Study published for:
Truly DEAD by intact cell wall containing DNa Compartments-?? I think that no such paper exists
This model is invlked by Bockenstedt to explain he "GLOBS of Borrelia" near the Murine
Lab infected Arthritsi model with huge Galaxy like numbers of supposedly nd allegedllyTotallly .
Dead borrelia
Counterpoint: Persisters - which at first glance APPEAR to be Dead- can be revived to
reproduce living borrelia

Extracellular RNa Degradation products
Intracelluar RNA ( foreign to the Host) - is there a kinetics of Degradation of RNA-- contained inside
either totallly Dead Borrelia containing RNA compartments? I think that no such paper exists.
If RNA is not degraded when present inside of the Carcases of Allegedly DEAD borrelia
then detection of such RNA cannot be used as proof of Viability.

Many variables about Kinetics need to be considered and published before conclusions and dogmatic
statements can be made on this forum.
Alan B.MacDonald, MD, FCAP, FASCP
Feb 2, 2014

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