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

Post by admin » Wed 10 Dec 2014 13:11

38 posts moved from this topic to a new topic Definition of late-stage Lyme disease because the discussion wandered off the subject of this thread. Please either keep off-topic comments limited or start a new topic.

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

Post by velvetmagnetta » Sat 13 Dec 2014 5:49

I have not been feeling well, so was not able to continue the conversation about BSK medium. When I get that ill, my body hurts a great deal, and the pain is just too distracting for me to accomplish much mental activity. At the same time my body gets beat up, so, too, does my mind - so trying to think through the quagmire that is my brain in those times is futile.

But now it all seems to be receding for a bit, so I can get back to the question of the lost art of Borrelia burgdorferi culturing (the "Gold-Standard" of Lyme disease diagnosis).

Because this conversation began several pages ago in this thread, I will quote Henry's full post:
Henry wrote:Velvetmagnetta: Here's the Barbour paper noting the ability to culture Borrelia on BSK starting from a single cell:
http://www.ncbi.nlm.nih.gov/pmc/article ... 0-0076.pdf . The statement is made at the bottom of page 522.

Barbour also notes that some of the ingredients used to prepare BSK -- especially the rabbit serum-- vary from batch to batch. Therefore the comparisons made in Figure 1 of the Sapi paper don't really help to determine if the Sapi modification is superior to BSK. What must be done is to use what is considered to be the optimal conditions for each, using the SAME batches of ingredients to prepare both types of media. Then, culture the same clinical specimens -- side by side-- using both types of media. Since they haven't shown the results of such an experiment, it is impossible to say that the Sapi modification represents a significant improvement over conventional BSK. To be perfectly fair, it would be best for a neutral third party to conduct such an experiment. That would settle the question. Aside from issues related to contamination, I predict there would be no significant difference -- or a slight difference at best, not worth paying the $500-$1,000 to have the test done by ALS, when another laboratory can do the test "for pennies" using conventional BSK.

A distinction should between: (a) the ability of BSK to culture Borrelia starting from a few or a single cells; and (b) numbers of Borrelia likely to be present in the blood (bacteremia) during various stages of infection. The former has been addressed by Barbour and others early in their work by doing limiting dilution assays; such quantitative studies were essential to establish the optimal conditions for culture, as well as to assess many of the variables involved. The latter became evident when BSK was first applied for use in clinical studies. It was then found that the bacteremia associated with Lyme disease is very transient. That is the reason why Wormser et al. were able to find only 48% of the patients they examined to be culture positive. It has little --if anything-- to do with the ability of BSK to support the growth of Borrelia. For all we know, the same percentage of positives (48%) might have been obtained if the Sapi modification had been used instead of BSK in that study. The results have to do mainly with the NUMBERS of Borrelia present in blood at the time of culture. Because Borrelia have at least 6-8 different kinds of adhesins/integrins on their cell surface, they are quickly taken out of circulation from the blood as they bind -- very tenaciously-- to matrix tissue. That is a well-known and characteristic feature of early infection in humans, not in mice. In view of these considerations, do you really think developing a better culture medium is going to help much, when that really isn't the main issue? Certainly, there is room for improvement in any laboratory test; however, I would not hold my breath for too long in this case -- in view of the aforementioned considerations.

In the above cited Barbour paper, he also notes that it is not unusual for Borrelia to form aggregates in liquid culture. These are what Sapi and others (MacDonald) incorrectly call biofilms. If you consult any good text book on Bacteriology and see what they have to say about biofilms, you will soon realize that what Sapi and others call biofilms in no way resembles the classical definition. What they see are laboratory artifacts, i.e., aggregates. But, as I said before, Sapi et al. are amateur bacteriologists.......
Thank you so much, Henry, for taking the time to find that reference! I very much appreciate it.

Indeed, Barbour does claim to be able to culture Bb from a single organism. From the 1984 Barbour paper:
...we have found in our laboratory that only the complete medium suffices for reliable achievement of spirochete densities of 1-4X10^8 per ml, generation times of approximately 11-12 hours, and growth of cultures from inocula of one to two organisms...


To be clear, Lorima pointed out that these spirochetes were obtained directly from tick mid-guts.

So, then, I had to re-read Dr. Wormser's Bb culturing efforts. Here is Wormser's effort from 2000 and a review from 2001:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC86513/
Comparison of the Yields of Blood Cultures Using Serum or Plasma from Patients with Early Lyme Disease
Gary P. Wormser,1,* Susan Bittker,1 Denise Cooper,1 John Nowakowski,1 Robert B. Nadelman,1 and Charles Pavia1,2

Abstract

In an initial experiment, culture-grown Borrelia burgdorferi was added to freshly collected uninfected human blood. This in vitro study demonstrated that more spirochetes were distributed into the plasma than into the serum fraction. In a subsequent clinical study, B. burgdorferi was recovered from plasma cultures of approximately 50% of 42 patients with early Lyme disease associated with erythema migrans. The rate of recovery from plasma cultures was significantly greater than that from serum cultures (P < 0.001).
So, he found the spirochetes tend to accumulate in the plasma fraction of the blood samples as opposed to the serum fraction. I think the idea is that Bb either prefers coagulants or Bb gets stuck to coagulants. Either way, he found lots more Bb in the plasma versus the serum. So, then, putting this new-found result to the test, he attempted to grow spirochetes from recently infected, EM rash, patient blood plasma.


The review:

http://jid.oxfordjournals.org/content/184/8/1070.full
Yield of Large-Volume Blood Cultures in Patients with Early Lyme Disease

Gary P. Wormser1,
Susan Bittker1,
Denise Cooper1,
John Nowakowski1,
Robert B. Nadelman1 and
Charles Pavia1,2


To improve yield, 6 3-mL plasma cultures (18 mL total) were established for adult patients with early Lyme disease associated with erythema migrans. Borrelia burgdorferi was recovered from the blood of 22 (44.0%) of 50 evaluable patients. The recovery rate per plasma culture and the frequency of positive results for plasma cultures for individual patients were consistent with a level of spirochetemia of ∼0.1 cultivable cell/mL of whole blood. Our findings suggest that, if further improvements in the yield of blood cultures are possible, they probably will depend on enhancing the sensitivity of the culture method rather than increasing the volume of material cultured
So, what Wormser found was that the frequency of positive results from early infection cultures was consistent with "a level of spirochetemia of ∼0.1 cultivable cell/mL". I am unsure if he means that this is all one can expect to find in newly infected blood, or if he means that was all they could find in this particular experiment.

The next sentence seems to suggest the latter, "Our findings suggest that, if further improvements in the yield of blood cultures are possible, they probably will depend on enhancing the sensitivity of the culture method rather than increasing the volume of material cultured" Here, Wormser seems to be stating that, yes, the spirochetes are there and it is the fault of the BSK medium why they cannot be grown from these early-infection blood samples.

Can anyone clear this up for me?

He seems to be saying there is definite room for improvement in the culture method, but why, if the BSK medium is already sufficient to grow an entire flourishing colony of Bb spirochetes from just one organism?

So, I don't know, Henry. You're saying that if spirochetes do not grow in BSK medium, then that means that the blood sample was devoid of any spirochetes. But that does not seem to be what Dr. Wormser is saying in his reports.

Now, I do see what Henry is saying about comparing BSK modification results side-by-side in a careful, incremental, quantitative way. Great idea. Somebody should do that at some point. Sapi did do it, albeit on a smaller scale - she compared 3 different modifications side-by-side and took the best one and ran with it.

(Henry, I encourage you to analyze that graph in Figure 1 of her paper. It looks simple, but there is a lot of information packed into that graph, including an impressive and statistically significant growth improvement.)

She then went ahead and grew those spirochetes on the best version of the BSK medium she could devise.

Henry said that culturing from BSK is easy and cheap. Everybody says that culturing Bb would be the Gold Standard of diagnosis, so why isn't it done?


As for biofilms...

I'm pretty sure Sapi knows the difference between an aggregation of spirochetes and an actual bio-film such as that which grows on teeth. A bio-film is made up of many different organisms that use the unique structures created by different species of the bio-film for protection against any outside threats. Bio-films are real structures found anywhere slime can grow and accumulate. That one sure doesn't take a microbiology degree to understand! Henry, please stop trying to put Dr. Sapi down. She's a very bright woman and we could all learn a lot from her.

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

Post by velvetmagnetta » Sat 13 Dec 2014 9:57

Re: uniform rabbit serum

Henry said:
Barbour also notes that some of the ingredients used to prepare BSK -- especially the rabbit serum-- vary from batch to batch. Therefore the comparisons made in Figure 1 of the Sapi paper don't really help to determine if the Sapi modification is superior to BSK. What must be done is to use what is considered to be the optimal conditions for each, using the SAME batches of ingredients to prepare both types of media. Then, culture the same clinical specimens -- side by side-- using both types of media. Since they haven't shown the results of such an experiment, it is impossible to say that the Sapi modification represents a significant improvement over conventional BSK.
Again, from the Sapi paper under "Materials and Methods"
Borrelia strains were maintained in complete BSK-H media, which includes 6% rabbit serum (complete media from Sigma-Aldrich #B8291) at 34oC with 5% CO2 and 100% humidity.
Sapi obtained her rabbit serum from Sigma-Aldrich, specifically #B8291. You can read about the ingredients and ratios and uses here on the vendor's website:

http://www.sigmaaldrich.com/catalog/pro ... &region=US

Three different concentrations of rabbit serum were tested: 6%, 12%, and 20%. There are no other batch numbers listed, so it looks like Sapi did, indeed, use rabbit serum from the same batch for each comparison.

It looks like every issue Henry has brought up criticizing the Sapi paper has been answered in the paper itself. If only Henry would read said paper, I am confident it would put his mind at rest.

velvetmagnetta
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Joined: Sun 23 Feb 2014 22:47

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

Post by velvetmagnetta » Thu 18 Dec 2014 1:11

Hmmm...Strangely silent. Is it the Carl Tuttle petition? Or maybe, perhaps, the potential signing of the Lyme bill by Cuomo?

Whatever it is, it looks like there may finally be a sea change in the direction of research of Lyme disease and in the treatment of patients who are chronically ill due to a Lyme infection. Whether the actual infection is chronic remains to be seen, but at least now maybe we'll be trying to figure that out!

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