Genovars of Borrelia in the Wild - great diversity

[inmacdonald]

Diverse Borrelia Genovars confirmed in small animal hosts in the Borrelia/tick/ small mammal equation:

Link:
http://www.plosone.org/article/fetchObj ... tation=PDF


Genomic Flux: A genome which is capriciously re-arranged in the wild;
Leading to diverse GENOVARs - leading to DIVERSE BORRELIA Proteins - Leading to DIVERSE Antibodies to Borrelia
in chronically infected mammals (including small mammal reservoir species and the large animal/humans who
are the Ultimate ( last host) parasitized life form.
A moving target of ongoing Genetic Variation...
Leading to co-existence of Diverse Borrelia Genovars
Resulting in DIAGNOSTIC FAILURE to match all possible GENOvars of Borrelia proteins with
One static Protein Test strip or ELISA :
_______________________________________________________________

Why does Serology Testing using B31 derived Test kits produce inconsistent testing results in
mammals chronically infected with Borrelia?
__________________________________________________________________

Plos ONE has an answer to this perplexing conundrum:

Link:
http://www.plosone.org/article/fetchObj ... tation=PDF

__________________________________________

Questions and Answers from this Research::
How many Borrelia different GENOVARS can co-exist in one small reservoir Mammal?
How many Borrelia different GENOVARS can be SIMULTANEOSLY TRANSMITTED BY A SINGLE TICK BITE
TO A SMALL MAMMAL or from the blood of a SMALL MAMMAL to Man?


CLUE: Correct answer to 1. to 2. > 33 unique Genovars can simultaneously exist for locus RplB,
80 unique genovars can simultaneously exist ( among 14 groups) for locusOspC
in small animal reservoirs of Borrelia speciesl
How many of this diverse borrelia genovar" BUFFET" multitude can be Transmitted by a Single tick bite to the Human ( ultmate host) animal.??
Clue: A whole Lot N>>>1 genovar, >>>2 genovars, >>>3
genovars of borrelia.

______________________Serology fails because of too many Genovars of borrelia in our Ecosystem
to be adequately resolved with a single genostrain [ B31 borrelia ]

_________________________________That is Why any Serodiagnostic Testing - IS DOOMED TO FAILURE !!
Too many borrelia genovars simultaneously present in Warm blood


“This study is coming from authors who are well known to express extreme views about Lyme disease, “ Lantos says. “I would look at it skeptically until it is peer reviewed.”

[/quote]
_____________________


Respectfully,
Alan B. MacDonald , MD FCAP FASCP
Feb 16, 2014

[LHCTom]

Serology fails because of too many Genovars of borrelia in our Ecosystem
to be adequately resolved with a single genostrain [ B31 borrelia ]

That is Why any Serodiagnostic Testing - IS DOOMED TO FAILURE !!
Too many borrelia genovars simultaneously present in Warm blood

Serodiagnostic testing based on a single B31 strain was doomed before this discovery but for essentially the same reason. This discovery further aggravates a basic problem of epitope <-> paratope binding failure due to genetic variation in the surface antigens. The 80 variations in 14 OSPC types is just the tip of an iceburg.

All the surface antigens are constructed by genes that code for specific amino acid sequences. Small changes in the codons that define the sequences or large ones due to plasmid horizontal transfer cause the surface antigen amino acid sequence to vary.

http://www.ncbi.nlm.nih.gov/books/NBK2396/

The surfaces of parasite molecules contain many overlapping antibody-binding sites (epitopes). An antibody bound to an epitope covers about 15 amino acids on the surface of a parasite molecule. However, only about 5 of the parasite's amino acids contribute to the binding energy. A change in any of those 5 key amino acids can greatly reduce the strength of antibody binding.

http://www.ncbi.nlm.nih.gov/books/NBK2394/pdf/TOC.pdf

The binding strength between a B31 derived antibody paratope and an altered amino acid sequence genetic variant strain is based on as a few as 5 amino acids and protein folding. A minor change in the codons of the gene that codes for the antigen for those 5 amino acids can eliminate the binding between the B31 antigens used in the Western Blot and the antibodies in the blood being tested. For OSPC, there are numerous variants that alter this binding. Variations in the codons that produce the amino acid sequences critical to epitope <-> paratope binding for any surface antigen will lead to the Western Blot showing NO or possibly weak bands for the altered antigen.

Naive B cells make IgM antibodies that typically bind with low affinity to epitopes. A particular epitope stimulates division of B cells with relatively higher-affinity IgM antibodies for the epitope. As the stimulated B cell clones divide rapidly, they also mutate their antibody-binding regions at a high rate. Mutant lineages that bind with higher affinity to the target antigen divide more rapidly and outcompete weaker-binding lineages. This mutation and selection produces high-affinity antibodies, typically of type IgA or IgG.

Even Gary Wormser et al admit

A potential concern with any serologic test to detect antibodies to Borrelia burgdorferi is whether the epitopes incorporated in the test provide sufficient cross-reactivity to detect infection with all of the pathogenic strains of the species.

in

http://www.ncbi.nlm.nih.gov/pubmed/18724824

Effect of Borrelia burgdorferi genotype on the sensitivity of C6 and 2-tier testing in North American patients with culture-confirmed Lyme disease.
Abstract
BACKGROUND: A potential concern with any serologic test to detect antibodies to Borrelia burgdorferi is whether the epitopes incorporated in the test provide sufficient cross-reactivity to detect infection with all of the pathogenic strains of the species. This is a particular concern for the C6 test, which is based on reactivity to a single peptide.
There was also a trend for the sensitivity of 2-tier testing to vary according to the ospC genotype for the 14 genotypes represented in the study

In the same abstract they hint at the basis for the problem

There was also a trend for the sensitivity of 2-tier testing to vary according to the ospC genotype for the 14 genotypes represented in the study

In the infamous CDC 2 tiered Western Blot confirmation, 10 surface antigens were chosen because Dressler found these most frequently in his small study in a single clinic which almost certainly saw patients with limited genetic variation from a single local geographic region. These patients were almost certainly infected with strains of Borrelia burgdorferi which had limited genetic variation from the B31 strain. A larger geographically diverse study would have shown the need to both increase the number of surface antigens used and reduce the ratio below 5/10. The CDC 2 tiered test is very good at finding infections in patients lucky enough to have been infected by a strain with limited genetic variation in its surface antigens.

This is essentially why only the US uses the 5/10 ratio and why most other countries have found the need to use local strains plus reduce the ratio below 5/10 to avoid this strain senstivity due to genetic diversity of the surface antigen genes. There are many other problems with the CDC 2 tiered test implementation but this genetic variants of the surface antigens doomed it in theory long before the other problems compounded things. This is the basis of why antigenic variation fools the immune system. It keeps the paratope <-> epitope binding a moving target. So does normal genetic variation which is put on steroids by horizontal gene transfer.

[Claudia]

Pamela Weintraub, in Cure Unknown, pgs 118-119, wrote:

Even Andrew Levin, founder and president of Immunetics, the Cambridge, Massachusetts, company that manufactures the Western blots used by commercial labs, has his doubts. Suspecting the CDC pattern was overly ridged and prone to miss cases, he'd obtained samples of validated patient blood and had them analyzed by a computer program built with help from the scientists at the Massachusetts Institute of Technology. Using a small NIH grant to develop his system, Levin showed the presence of several antibody patterns, of which the CDC's was just one - and not the best one, at that. "If the two-tier criteria had been the best possible criteria," notes Levin, the computer program "would have taken us to it. But it did not. A number of other patterns emerged as the statistical front runners, instead." *

Surveying the limitations of the tests, Stony Brook scientists suggested the best way to diagnose Lyme disease might be to look not at a single test, but changes in sequential tests over time. They saw signs of positivity in rising antibody levels over subsequent ELISA's or the flowering of bands on Western blots conducted over months. But it was the rare pediatrician or family practitioner who understood these concepts and could follow suit. Instead, it was the CDC formula that stuck.

* In-person interview with Andrew Levin, Cambridge, MA, 2003

[LHCTom]

Even Andrew Levin, founder and president of Immunetics, the Cambridge, Massachusetts, company that manufactures the Western blots used by commercial labs, has his doubts. Suspecting the CDC pattern was overly ridged and prone to miss cases, he'd obtained samples of validated patient blood and had them analyzed by a computer program built with help from the scientists at the Massachusetts Institute of Technology. Using a small NIH grant to develop his system, Levin showed the presence of several antibody patterns, of which the CDC's was just one - and not the best one, at that. "If the two-tier criteria had been the best possible criteria," notes Levin, the computer program "would have taken us to it. But it did not. A number of other patterns emerged as the statistical front runners, instead." *

There is no such thing as the "best" antibody pattern in the context of infections throughout the entire US. Every study selects a particular cohort of patients with a particular mix of Bb strains and even occasionally species. The break between a strain and species of a bacteria such as Borrelia has evolved and now with 8 houskeeping gene Multi-locus PCR, has been redefined by a numerical genetic difference. That means that near the numerical boundary between a strain and species, a single polymorphism can exceed the numerical value and its a new species. This means you can have Borrelia burgdorferi strains that are quite genetically different from say B31 and very close to B. bissettii, B. kurtenbachii, B. americana, B. andersonii etc.. or other species now now seen in the US occasionally.

Becuase every cohort of patients is infected with different strains and possibly multiple strains, a "best" antibody pattern can only be calculated statistically for that cohort based on that mix. It means very little relative to evrey infection in every state by every strain or species in the US. It only works for that limited set of strains in the study. If one was to do an exhaustive study with thousands of patients in every endemic state, and the B31 derived antibodies were used in the ELISA and Western Blot, it is likely that NO useful pattern would be found. As the study contains more and more Borrelia that have varied surface antigen genes, in the limit, no useful pattern would emerge. This is because the B31 has specific surface antigen genes with specific amino acid sequences with paratopes very finley tuned to bind to sets of about 5 amino acid sequences. If the genetic variations change to codons that code for these 5 amino acids or effect the surface antigen folding, all bets are off.

As the surface antigen genes diverge genetically from this B31 strain, eventually none of the B31 paratopes would bind to the antibodies in patients with the divergent surface antigen genes where the key stes of amino acid sequences had changed. This is why the test rarely if ever catches different species. But remember a different species can be closer genetically to a Bb strain than to the B31 since the changes acrue as one nucleotide at a time or even jumps due to horizontal gene transfer. The changes that effect the surface antigen genes have the largest inpact on this problem. So the only way to make a test like the Western Blot uniform across all encountered US genotypes would involve using antigens derived from a "good" and "balanced" mix of genotypes from across the US chosen with some knowledge of paratope <-> epitope binding. Some of the Early 90 studies understood this problem and used mixes of as many as ten strain antigens. The selection of the "best" ten is a complicated task involving analysis of paratope/epitope binding sites but just providing a good mix based on known OSPC variants and geographically varied strains would probably perform much better.

This is the underlying reason Igenex and Stonybrook chose 2 strain antigens as their Western Blot antigens rather than the single B31. But this is not really adequate yet does appear to perform better in the real world of genetically diverse Borrelia. But even the idealized mix of antigens which minimized the paratope <-> epitope binding problem would probably still not be sufficient due to the many other issues associated with the temporal human immune response combined with varied up and own regulation of surface antigens over time in vivo. Then there are the bound versus free antibody problem and the lab to lab variations and difficulty interpreting a Western Blot etc...

Its a doomed approach. This is an engineering problem, not a science, math or medical problem. Its Bad engineering by those not educated in engineering.

[Claudia]

Agree. Thanks for summarizing -- and explaining -- the big picture so well. The quote from Levin was to point out how problematic the CDC 10 band selected criteria is, clearly illustrated by running a computer program to analyze band patterns just on his samples (maybe even supplied to Levin from the NIH themselves to implement the research grant proposal).

[Martian]

Am I the only one who thinks many of inmacdonald's posts are a mess, hardly readable, and hardly if at all acceptable, especially in the "Science" section? (e.g. the opening post of this topic)

Can anyone tell from his inmacdonald's post where this is coming from:

“This study is coming from authors who are well known to express extreme views about Lyme disease, “ Lantos says. “I would look at it skeptically until it is peer reviewed.”

[TwiceBitten]

Can anyone tell from his inmacdonald's post where this is coming from:

“This study is coming from authors who are well known to express extreme views about Lyme disease, “ Lantos says. “I would look at it skeptically until it is peer reviewed.”

webcache.googleusercontent.com/search?q=cache:http://www.outsideonline.com/fitness/bo ... -STD-.html

[Martian]

TwiceBitten,

Yes, but I did not ask for the source. I asked if one could tell the source from inmacdonald's post.

[TwiceBitten]

Can anyone tell from his inmacdonald's post where this is coming from:

“This study is coming from authors who are well known to express extreme views about Lyme disease, “ Lantos says. “I would look at it skeptically until it is peer reviewed.”

No.

[lou]

So doesn't this mean that antibody testing is doomed, can never work to catch all cases.....that antigen testing is the way to go? And will direct testing also have problems because of the issues you have identified?