Why aren't persisting spirochetes enough evidence of infecti

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inmacdonald
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Why aren't persisting spirochetes enough evidence of infecti

Post by inmacdonald » Sun 25 Nov 2012 17:29

Images
Borrelia burgdorferi cultured from human blood
Laboratory : Advanced Laboratories
Announcement: January 2012 bulletin
Patient population: Chronic Lyme Disease patients - many of whom
completed multiple courses of antibiotic therapies - but did
not experience relief of their symptoms of Chronic Lyme disease
Quality Control: All borrelia burgodrferi isolates -pure culture
All isolates immuno reactive with Monoclonal antibodies
which uniquely react with Borrelia burgodrferi
and which do not cross react with Treponema pallidum,
or relapsing fever grooup Borrelia [ie B. hersmii, b.miyamotoi etc}
Molecular Studies of positive isolates: DNA sequencing of all recovered strans completed
to assure their classification as Borrelia burdorferi.
No two strains identical to each other (molecular variation in
DNA bases -single nucleotide polymorphisms detected among theisolates.
What do the Borrelia grown from the blood of patients wthe Chronic Lyme Diseaxe-
antibiotic treatment Refractory type actually look like under the microscope?
[ See Images below from the Advanced Labs Buletine Jan 2012]
Images of Cutured borrelia burgdorferi from Human Blood.jpg
Borrelia burgdorferi recovered in blood culture - Chronic Lyme Disease patients
Images of Cutured borrelia burgdorferi from Human Blood.jpg (83.01 KiB) Viewed 2900 times
Submitted November 25,2102
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dorothy de kok
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Re: Why aren't persisting spirochetes enough evidence of inf

Post by dorothy de kok » Mon 26 Nov 2012 16:23

Well done to Advanced Laboratory Services. This is exactly what is needed!
It is just a pity we do not have access to ALS services in South Africa. But the positive effects ie. awareness of the chronic nature of LD, will definitely trickle through to the rest of the world.

I was also pleased to see that not all Bb spiro's are textbook corkscrews.

Thanks for posting this, Alan.

Dorothy

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inmacdonald
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Re: Why aren't persisting spirochetes enough evidence of inf

Post by inmacdonald » Mon 26 Nov 2012 17:37

Dear Dorothy,


It is my experience and the experience of others
that in initial cultures from human to the Lab environment,
that the borrelia first visualized are short in length and
usually not textbook corkscrews.

The process of adaptation from whatever form the borrrelia
assume in the solid tissues or in the blood
possibly involves Shape shifting from a non-corkscrew form
to an uncoiled form ( sort) to progresiveely longer length forms
to spiral forms.

We have had many discussions about the so called lack of proof
for the existence of Biofilms of Borrelia burgdorferi. You will note
that such discussions have ceased with the publication of our PLOS ONE
article on In Vitro Biofilm formation by borrelia burgdorferi.

We have had many discussions about the non- existence of Shape shifted
Borrelia forms ( ie cystic forms and granular forms and Cellwall deficient forms)
You will note that those discussions continue to this day.

We have had many discussions about the issue of Chronic Lyme Disease cohorts
of patients who note a variety of signs and symptoms. There has been, heretofore,
a detente between the NIHILISTS on the veracity of Chronic living borrelia inthe bodies of such patients,
and those who might be called the PLAUSIBILISTS.

The Images in this post, which are just an introduction to the full collection of
blood culture positive borrelia recovered from cultures of blood of patients who
experience Chronic Lyme borreliosis and who have undergone long term antibiotic treatment
without cure of their illnesses prove that Chronic Lyme borreliosis is Chronic because the
Borrelia are still alive in their Blood and elsewhere in their bodies.


Best wishes to you in South Africa, where for the moment,the NIHILISTS are still holding control,
alan

dorothy de kok
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Re: Why aren't persisting spirochetes enough evidence of inf

Post by dorothy de kok » Mon 26 Nov 2012 18:10

Thanks for that run down, Alan.

The NIHILISTS may currently have control here, but it will change...

RobertF
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Re: Why aren't persisting spirochetes enough evidence of inf

Post by RobertF » Mon 26 Nov 2012 22:09

Dear Alen, you dont have to convince us but them

Seven years ago I was at the congres of the LDA in England and met Andy Wright and Maria Kroun. Both very experienced in micrsocopy We had an afternoon session about darkfield microscopy. As they requested for volutiers I was the first in the row. Living spiro were seen or what we saw looked like spiro, but still has to be confirmed to be Borrelia.
I was so excited that I bought myself a Leitz microscope with condensor darkfield and after some trial and error I had many nice samples of my own blood. There is no doudt that they are there, when pressing the slide even more appear, but objectively seen you have to confirm it by biochemical or Immuno or PCR tests that what you see is a Borrelia.
I think that this test works therefor whith immunofluorescence technic.

The problem is that you cannot automate it for routine use.
The next problem is cost. An Elisa cost about 10 euro and the insurance pays this. They will not pay for the Spirofind which cost 217 euro even if it would have a sensitivity of 100%

This microscopic test is very usefull in the beginning of the disease. I have never understood how MD's ethical accept that they send an infected person home and tell him/her to return in 6 weeks because the miserable Elisa cannot be used prior. It is a ridiculous situation because we all know the sooner you treat the greater the change of cure.
What we need is a reliable test for the next day after a tickbite
So how do we convince them!!

Remark, acc to Barthold you cannot culture Borrelia after antibiotic treatment. Will the 4 weeks be enough?

Lorima
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Re: Why aren't persisting spirochetes enough evidence of inf

Post by Lorima » Tue 27 Nov 2012 16:17

Hi all, 

I'm inclined to apologize for another long post. I think I'll start a thread asking if people would prefer I try to make them shorter, in Lyme Cafe. For now, I'll go ahead and post the thing. It just takes too long to cut these things down to size without either losing relevant (IMHO) points, or obscuring the chain of thought that connects them. 

Thanks, Dr. M, for sharing this info. I agree that this method should be adequate proof, from a scientific standpoint. It may (probably?) be blocked due to political considerations, if it shows any signs of being able to detect LATE LD patients. Seems unlikely to be useful for that, though, because presence of Bb in the blood is so scanty and intermitent, in late disease. 

From a scientific standpoint, I think Bb-specific IgM antibodies or PCR from blood or urine should also be acceptable, to justify treatment. But they are not, according to IDSA guidelines, and CDC/FDA warnings about "unvalidated" tests. I suspect the putative high-background issues with IgM could be overcome by good technical means, and already has been, at specialty labs like Igenex. But in mainstream medicine, it has been moved off the table politically, by CDC, IDSA, insurance companies, and EUCALB saying that IgM evidence is invalid in late disease. PCR is dismissed as lab contamination, or dead bacterial remains, or just as "unvalidated". 

I'm not sure what they'll say about any new test; do they really WANT to detect and treat more LD cases? Could they be dragged kicking and screaming into allowing more detection and treatment of LATE LD? So far it seems not. The active campaign against "unvalidated" tests that are scientifically sound, makes me think they are actively trying to prevent more diagnosis. By saying that in all stages, the bacteria can be eradicated easily, and that late complications are rare, they are further discouraging the development and acceptance of new tests by mainstream medicine. 

Even so, it's nice to have culture available commercially, for LLMDs to order for patients if they want it, can afford it, and have reason to think there will be sufficient Bb in the blood to detect it. It's a very slow test, though. 

Robert, what you say above, sounds right to me. Thanks for writing. 

Aside from Barthold's culture comment ("Remark, acc to Barthold you cannot culture Borrelia after antibiotic treatment. Will the 4 weeks be enough?"),
I'm wondering if it's likely that even untreated late Lyme patients who are stable (though not healthy) ever have enough bacteria in their blood at one time, to succeed at this, OR at very sensitive PCR. We don't really know - but the model dominant among, say, ILADS physicians, is that there are intermittent releases of bacteria into the blood from the colonies/biofilms in the tissues, and that seems biologically the most plausible, to me. How much bacteria? How often? No one knows, and it's probably unique to each case. 

How would one target the timing of the blood draw to coincide with that? 
Maybe choose for a study patients who have possible signs of recurrent systemic spirochatemia, like fevers and/or night sweats? My family patients have had episodes of that, but not often. Should we think about  analogies with the relapsing fever borrelia, and see how their fevers relate temporally to their bursts of spirochetemia? 

I wonder, along with Robert, if waiting 4 weeks after stopping Abx is long enough. It seems the bugs would just be waking up. If they wake up randomly and sporadically after Abx removal, as the scout hypothesis suggests (http://www.lymeneteurope.org/forum/view ... f=5&t=4313 ), there's not going to be a sudden big burst of spirochetes in the blood. 

But if there were an obvious exacerbation of symptoms suggesting spirochetemia at the 4 week mark, in particular patients, it might be worth a try. This test is so expensive, a patient really wouldn't want to try multiple times at random, hoping to hit the timing just right. 
The company does say that "active disease" is more likely to yield results; it would be good to narrow that down and be more specific about both relevent symptoms, and typical timing of symptoms with spirochetemia. 

I wonder how this culture sensitivity compares to PCR? Theoretically, you could detect as litte as one organism by either method, so you'd have to compare them pragmatically side by side, with people who are gifted in each technique doing them side by side on the same samples. PCR is overwhelmingly faster and cheaper. But how sensitive you can get it to be, is an empirical question, for both techniques.  

I looked in the literature for PCR improvements, and here's a gem. It's being validated on early patients that can be proved to meet CDC case requirements, so far. The involvement of Abbott (huge pharma) might give it a chance of commercial success. Notice Schutzer and Aucott, rare mainstream researchers who get it, that the current LD situation is a big problem.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3348129/
PLoS One. 2012;7(5):e36825. Epub 2012 May 8.
Direct molecular detection and genotyping of Borrelia burgdorferi from whole blood of patients with early Lyme disease.
Eshoo MW, Crowder CC, Rebman AW, Rounds MA, Matthews HE, Picuri JM, Soloski MJ, Ecker DJ, Schutzer SE, Aucott JN.
Source
Ibis Biosciences Inc, an Abbott Company, Carlsbad, California, United States of America. mark.eshoo@abbott.com
Abstract
Direct molecular tests in blood for early Lyme disease can be insensitive due to low amount of circulating Borrelia burgdorferi DNA. To address this challenge, we have developed a sensitive strategy to both detect and genotype B. burgdorferi directly from whole blood collected during the initial patient visit. This strategy improved sensitivity by employing 1.25 mL of whole blood, a novel pre-enrichment of the entire specimen extract for Borrelia DNA prior to a multi-locus PCR and electrospray ionization mass spectrometry detection assay. We evaluated the assay on blood collected at the initial presentation from 21 endemic area patients who had both physician-diagnosed erythema migrans (EM) and positive two-tiered serology either at the initial visit or at a follow-up visit after three weeks of antibiotic therapy. Results of this DNA analysis showed detection of B. burgdorferi in 13 of 21 patients (62%). In most cases the new assay also provided the B. burgdorferi genotype. The combined results of our direct detection assay with initial physician visit serology resulted in the detection of early Lyme disease in 19 of 21 (90%) of patients at the initial visit. In 5 of 21 cases we demonstrate the ability to detect B. burgdorferi in early Lyme disease directly from whole blood specimens prior to seroconversion.
PMID: 22590620 [PubMed - indexed for MEDLINE] PMCID: PMC3348129 Free PMC Article
Excerpts:

snip
Improved PCR/ESI-MS sensitivity by Isothermal Amplification

To detect the low levels of Borrelia genomic DNA in whole blood samples from early Lyme disease patients we developed an isothermal amplification (IA) assay that amplifies the seven B. burgdorferi target regions used with a previously described Borrelia broad-range PCR electrospray ionization mass spectrometry detection and genotyping assay (PCR/ESI-MS)[16]. The strategy behind this approach uses a combination of a strand-displacing DNA polymerase in conjunction with multiple primers to selectively amplify the target region in an isothemeral reaction. The use of multiple primers flanking the target sequence allows outer primers to displace the DNA strands created by inner primers allowing the newly displaced strand to function as a template for further amplification in a manner similar to multiple displacement amplification [18]. The IA assay was designed to specifically work with the entire output of DNA extraction from 1.25 mL of whole EDTA blood to ensure amplification of low levels of Borrelia DNA that may be present in the specimens. The Borrelia enriched DNA can then be used directly in the PCR/ESI-MS assay without any purification. Since the IA is coupled with PCR/ESI-MS specificity for the target detection is maintained through the PCR/ESI-MS. Using extracts from 1.25 mL of human blood spiked with various amount of quantified B. burgdorferi DNA (Barbara J. Johnson CDC, Fort Collins, CO) (200 genome copies down to 0.3 genome copies) we compared the sensitivity of the PCR/ESI-MS assay to detect B. burgdorferi with and without the IA B. burgdorferi DNA enrichment (Figure 1). Detection was determined when one or more of the eight primers detected B. burgdorferi. As shown in Figure 1, samples that were not enriched (neat) reproducibly detected down to 200 B. burgdorferi genomes. When the IA B. burgdorferi DNA enrichment was employed the sensitivity was reproducibly improved down to 0.6 genomes. This demonstrated the IA B. burgdorferi DNA enrichment process resulted in over a 200-fold increase in sensitivity in the PCR compared to untreated.
Note Barbara Johnson at CDC cooperated; maybe that will help some, politically? She does seem amenable to improving diagnosis of EARLY cases. 

From the discussion (it's worth reading the whole section, but here's a snippet): 
Since the adoption of the current two-tier serologic based test for Lyme disease surveillance there have been few practical alternative diagnostic tests available to the clinician. The ability to culture B. burgdorferi has been important for use in research applications to confirm the diagnosis of B. burgdorferi infection, but is not available or practical for use in a clinical setting because it requires weeks before cultures become positive. Past attempts at PCR diagnosis for North American Lyme disease with blood samples has been largely unsuccessful due to the low sensitivity [3], [4]. European researchers have reported varying rates of success using PCR to detect members of the B. burgdorferi s.l. group from blood or its components; 7.5% up to 78.1% [26], [27], [28]. The difficulty of detecting Lyme Borrelia in the blood has been attributed to the low numbers of organisms circulating in the blood stream in acute infection [7], [14], [29]. In recent studies in North America, using larger blood volumes in addition to utilizing more sensitive nested PCR tests have had increased success in detecting the low densities of Borrelia that are typically present in the blood in early Lyme disease [6], [7]. These studies also demonstrated that a direct detection strategy can work provided enough blood is examined and the assay is sensitive down to genome copies numbers in the 10–100 range.

To overcome the challenge of extremely low density of spirochetes in the blood and increase assay sensitivity we utilized a combination of strategies. By using a relatively large volume of whole blood we increased the probability of B. burgdorferi DNA being present in the specimen. Next, the IA strategy developed here enriches the entire nucleic acid extract for Borrelia DNA prior to PCR thereby increasing the probability that any Borrelia DNA present is detected. Lastly we used a mass spectrometry-based multi-locus assay to increase the number of possible targets in the Borrelia genome we may detect. Extracts subjected to IA can also be used to screen for other pathogens without affecting the sensitivity of the individual assays (data not shown). All of the specimens in the study were tested for other tick-borne pathogens including Babesia, Anaplasma, Ehrlichia and no co-infections were found (data not shown). A key technical development of the isothermal amplification is that it uses the entire output of a whole blood DNA extraction and employs a set of 50 primers per target locus (350 primers total) to assure amplification of the B. burgdorferi target loci even the presence of high levels of human DNA. The isothermal amplification is a relatively simple addition to a PCR assay work flow and this approach could be applied to other assays where target DNA of interest may be present but at levels below the limits of detection by PCR. Furthermore the method should be applicable to DNA extracts from a variety of specimen types such as tissue, cerebrospinal fluid, serum, plasma or synovial fluid.
This is very good. Again, it's very cheap and fast compared to culture. I wonder if the CDC's current official acceptance of culture as proof of infection, would hold up in the face of a commercially available culture test. I rather doubt it; their collaborating with the FDA and IDSA in sending out letters "warning" physicians not to use "unvalidated" tests seems to indicate an active interest in preventing diagnosis by any means other than EM and the two-tier test interpreted by Dearborn criteria, or other serological assays that do NOT rock the boat by detecting more late patients. (This is ensured by using Dearborn as a gold-standard for defining "real" Lyme patients.) Their current rejection of commercially available PCR (which I assume was, like Dearborn, under the influence of Steere and collaborators as the world experts on the disease) could easily be extended to Advanced Lab's culture method, simply by calling it "unvalidated". (Please correct me if there's some unpolitical way to get a test "validated" by the CDC.) Apparently FDA approval isn't enough; Igenex is allowed by the FDA to market its services, but the CDC doesn't accept them, and somewhat actively campaigns against them. 
The CDC's most obstructionist LD researchers are nearing retirement age. But I assume Steere will use his influence to ensure that no reform-minded younger cohort will take their place. 

Robert, regarding microscopy, it's interesting that you (and Carina) could see organisms in your blood. That must mean there are a large number of them They would likely be detectable by PCR - did you ever have that done?
Can you still see them after treatment? Were there systemic symptoms that seemed to correlate with their increased presence?

Best wishes, 
Lorima
"I have to understand the world, you see."
Richard Feynman

dorothy de kok
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Re: Why aren't persisting spirochetes enough evidence of inf

Post by dorothy de kok » Wed 28 Nov 2012 9:54

Robert, regarding microscopy, it's interesting that you (and Carina) could see organisms in your blood. That must mean there are a large number of them They would likely be detectable by PCR - did you ever have that done?
Can you still see them after treatment? Were there systemic symptoms that seemed to correlate with their increased presence?
I was interested by this, as I too can see spirochetes under the microscope. After treatment there are few in the plasma, and then they are almost impossible to detect. But if you lower the objective onto the slide until the red blood cells are crushed, many spirochetes are released. We have to remember that borrelia is an intracellular organism, and that is where we need to look in order to find them.

I am attaching two files showing images of the spirochetes. The first one is compiled by Dr Alan MacDonald who very kindly included my images near the end of the PDF. THe second is another collection of images of the blood of a person diagnosed with schizophrenia who has recovered significantly while on treatment (three courses of antibiotics followed up with continuous anti-spirochetal herbs).

Yes, the spiro's are definitely still there after treatment and while on treatment. The numbers may be reduced slightly following treatment, but their presence is undeniable and they ARE EASY TO FIND. That is why I cannot understand how anyone can deny the existence of chronic Lyme. It is a no-brainer.

We have recently sent DNA samples to Europe for PCR. Hopefully the spiro's will soon have an identity.

https://www.dropbox.com/s/ywxbu11pt6cgf ... rchers.pdf

https://www.dropbox.com/s/p7zv9chuhe1ksvu/justin1.pptx

Many thanks

Dorothy

Lorima
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Re: Why aren't persisting spirochetes enough evidence of inf

Post by Lorima » Wed 28 Nov 2012 14:35

Very interesting! I'm eager to know what PCR shows about your sample.

Thanks Dr. M for posting the photos and information!

I bumped Carina's microscopy thread - it's in the Off-Topic section of Lyme Community, here:

http://www.lymeneteurope.org/forum/view ... f=8&t=3972
"I have to understand the world, you see."
Richard Feynman

dorothy de kok
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Re: Why aren't persisting spirochetes enough evidence of inf

Post by dorothy de kok » Wed 28 Nov 2012 15:52

I bumped Carina's microscopy thread - it's in the Off-Topic section of Lyme Community, here:
Thanks for the link, Lorima.

Lorima
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Re: Why aren't persisting spirochetes enough evidence of inf

Post by Lorima » Wed 5 Dec 2012 15:56

Bump, to keep Advanced Lab culture posts together.
"I have to understand the world, you see."
Richard Feynman

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