Eva Sapi study about new Borrelia blood culture test

Topics with information and discussion about published studies related to Lyme disease and other tick-borne diseases.
Martian
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Eva Sapi study about new Borrelia blood culture test

Post by Martian » Fri 7 Mar 2014 22:47

Source: http://www.ncbi.nlm.nih.gov/pubmed/23470960 (PubMed)
Int J Med Sci. 2013;10(4):362-76. doi: 10.7150/ijms.5698. Epub 2013 Feb 18.

Improved culture conditions for the growth and detection of Borrelia from human serum.

Sapi E, Pabbati N, Datar A, Davies EM, Rattelle A, Kuo BA.

Abstract

In this report we present a method to cultivate Borrelia spirochetes from human serum samples with high efficiency. This method incorporates improved sample collection, optimization of culture media and use of matrix protein.

The method was first optimized utilizing Borrelia laboratory strains, and later by demonstrating growth of Borrelia from sera from fifty seropositive Lyme disease patients followed by another cohort of 72 Lyme disease patients, all of whom satisfied the strict CDC surveillance case definition for Lyme disease.

The procedure resulted in positive cultures in 47% at 6 days and 94% at week 16. Negative controls included 48 cases. The positive identification of Borrelia was performed by immunostaining, PCR, and direct DNA sequencing.

KEYWORDS: Clinical microbiology, Lyme disease, Spirochetes., Vector-borne diseases

PMID: 23470960 [PubMed - indexed for MEDLINE] PMCID: PMC3590594
Full text:

http://www.medsci.org/v10p0362.htm (Int J Med Sci)
http://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23470960/ (PubMed Central)


Note: I know this was posted before, but those topics got messy IMO.

Camp Other
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Re: Eva Sapi study about new Borrelia blood culture test

Post by Camp Other » Fri 7 Mar 2014 23:57

Yes, they did get messy, didn't they: http://www.lymeneteurope.org/forum/view ... f=6&t=4545

Martian, is there some specific issue/idea you wanted to raise with reposting the above study?

Martian
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Re: Eva Sapi study about new Borrelia blood culture test

Post by Martian » Sat 8 Mar 2014 0:25

Camp Other wrote:Martian, is there some specific issue/idea you wanted to raise with reposting the above study?
I think that important/controversial studies deserve a properly formatted topic in the "Science" section with relevant information gathered and with on-topic substantial discussion in case it evolves.

Martian
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Re: Eva Sapi study about new Borrelia blood culture test

Post by Martian » Sat 8 Mar 2014 1:24

The lab that performs the Sapi blood culture test is Advanced Laboratory Services. Information about the test can be found on these pages:

Spirochete/Borrelia Testing (from Culture): http://www.advanced-lab.com/spirochete.php
Spirochete/Borrelia Testing: Frequently Asked Questions: http://www.advanced-lab.com/faq.php

Quote from the FAQ:
What is the cost for the test, and how may I pay for it?

There are several test options available for the Spirochete/Borrelia Culture. A more complete description of test options can be found here. The test prices are as follows:

Basic Borrelia Culture: $595
Comprehensive Borrelia Panel: $920
Monoclonal Borrelia Culture: $695
Comprehensive Monoclonal Borrelia Panel: $1020

Margherita
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Re: Eva Sapi study about new Borrelia blood culture test

Post by Margherita » Sat 8 Mar 2014 1:31

I think that important/controversial studies deserve a properly formatted topic in the "Science" section with relevant information gathered and with on-topic substantial discussion in case it evolves.
Well Martian, as there are lots of messy topics and controversial things with regard to Lyme disease, I'ld say:

BUON LAVORO!


You will surely run out of time. ;)

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LHCTom
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Re: Eva Sapi study about new Borrelia blood culture test

Post by LHCTom » Sat 8 Mar 2014 18:19

When I first read the study, I reviewed the phylogenetic tree carefully and was surprised to see the large number of B. garinii without so much as a comment in the paper. I thought at the time that this had many important implications. For example, its well known the B31 based antibody testing will not typically detect B garinii as was discovered in the 90's in Europe. That alone would have been big news, but the authors did not even mention it. All of the samples in the top half of the tree were B garinii. Its clearly shown and evident. I was also surprised that the table of polymorphic changes was only referenced to B burgdorferi B31 which seemed odd given half the samples begged for a B garinii comparison. Had another table compared polymorphic changes against B garinii, it would have clearly shown what was obvious from the phylogenetic tree. Either US researchers had missed B garinii being common in the US, or something was amiss.

http://www.medsci.org/ms/getimage.php?n ... type=thumb

I recall I was puzzled because I knew B garinii was routinely found in Europe and Asia but had never been seen in the US. It had been found once in ticks on seabirds from Gull Island, Newfoundland, but never in the US. This should have been a big red flag but I was so happy with the amazing results, I let my guard down. In addition to the new culture success, the ALS/Sapi group would have been the first to find actual human infections with B garinii in the US. This discovery would have been big Lyme ecology and epidemiology news. If it was true, it would have rippled everywhere.

Even if this study suffered from contamination, it doesn't mean the culture isn't good and useful. It just means the high sensitivity found in the study is meaningless and the whole study needs to be repeated with great care. Its a real shame because the errors in the study have given mainstream doctors a reason to distrust claims and concerns regarding Lyme. Its also a shame ALS has not said a word about it and even removed most of the culture information from their website. I know there are people who are unhappy when someone like me points out issues with the ALS culture study but I do believe most people really want the truth and be able to trust these crazy tests.

A culture is so obviously needed in the clinical setting but I'm afraid when the kinks are worked out, cultivable Borrelia will not be so easily found as has been seen with people taking the ALS culture test. I'm afraid those of us with chronic Lyme may have uncultivable Borrelia as has been seen in most of the animal studies. Lets hope ALS finishes their follow-up studies so we know how useful the culture really is. It would be a shame to give up now since the culture is by far the best way to study human Lyme infections if the uncultivable hypothesis is not correct.

Borrelia garinii in Seabird Ticks (Ixodes uriae), Atlantic Coast, North America
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3291351/
Although B. garinii is present in seabird ticks in a nearly circumpolar distribution in both the Northern and Southern Hemispheres (12,13), including Alaska, the presence of B. garinii in I. uriae ticks at sites on the North Atlantic Coast has not previously been documented. We sought to determine whether B. garinii is present in ticks obtained from colonial seabird nesting sites on the Atlantic Coast of North America.
Global ecology and epidemiology of Borrelia garinii spirochetes
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3426327/

Sapi Study Phylogentic Tree.jpg
Sapi Study Phylogentic Tree.jpg (31.96 KiB) Viewed 2641 times
The greater the ignorance, the greater the dogmatism.

Attributed to William Osler, 1902

nnecker
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Re: Eva Sapi study about new Borrelia blood culture test

Post by nnecker » Sat 8 Mar 2014 20:15

I would like to see more detail about the patients history,i.e whether or not they were ever treated when they were diagnosed for Lyme.

I would think that most of the patients would start abx's right away once they found out that they were seropositive for Lyme.If in fact this is the case,why can Sapi easily culture viable spirokeets after abx treatment when other researchers can never culture them( even though they use tissue samples as well ) in lab animals from mice to monkeys?

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LHCTom
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Re: Eva Sapi study about new Borrelia blood culture test

Post by LHCTom » Sun 9 Mar 2014 1:38

Here is a patient history that might be interesting. I was positive for IgG 18, 34, 39, 41, 58 and 66. I was also positive by the Immunetics C6 ELISA kit at IGenex and StonyBrook ranging from 1.43 - 1.87 where > 1.1 is positive. I was then tested positive by the ALS culture. I was put on 1 gram/day Ceftin and Tinizadole. After 3 months on the Abx and while still on the Abx, I was re-cultured positive. That surprised me given the Abx load in my blood. So I continued the Abx until about 8 months and was yet again re-cultured about 3 weeks after stopping the Abx. I was yet again positive. I ordered the sequencing option.

I scanned my pyrG sequence faxed to my doctor into a file and very carefully checked it nucleotide by nucleotide against the fax. The fax original came from Eurofins who did the sequencing for ALS. So the sequence data I received was Eurofins sequence data that did not appear to have been altered but copied and faxed. In the middle of the 603 nucleotide main pyrG sequence, there were 2 "x"s where it appeared the sequencing failed with x = unknown. I ignored those leaving 601 usable nucleotides to run on BLAST. After ignoring those 2 unknown nucleotides, I ran BLAST and was a 100% match to the same set of Bb where the pyrG matches B31 just like the 10 isolates from the Sapi/ALS submissions in table 3. Its possible I have a B31 strain but it seems a stretch given I live in CA. So that wasn't terribly definitive. But culturing positive while on Abx for 3 months and just after 7 months did make me wonder. That's just one real life patient history that occurred during the same time period while the ALS study was underway. I have to admit it really bothered me at the time.

I just had the new low temperature nested PCR test with sequencing done. They run 3 PCR tests per sample so I ordered it twice so they will run mine 6 times. Better odds. Even though I'm not terribly hopeful, if it shows positive, they will provide me the 293 nucleotide 16S ribosomal RNA gene. Then I would be able to see if it also matches the B31 strain (or not). I've not yet got the results. It will probably be negative but if not it will be interesting.

Why am I not hopeful? This PCR test in theory can detect a single spirochete. They take about 3ml of blood. The PCR test uses less than .1% of the entire sample. So if the test is run 3 times, the 3ml of blood will need to contain about 1000 spirochetes to find one in the final test sample unless of course one is lucky. If you think about it, 1000 spirochetes is quite a few per 3ml. There either needs to be a very effective concentration technique or you need to run many many tests to be sure there is at least one spirochete in the final PCR test sample.

One reason culture is so effective is not only can you culture the entire 3ml ( or more), but then they replicate exponentially so when the PCR is done, the number of spirochetes is quite high and the probability of finding at least one in the small PCR test sample is large. Its a numbers game.

http://www.dnalymetest.com/

Paper describing the test: http://ajcp.ascpjournals.org/content/13 ... l.pdf+html
Last edited by LHCTom on Sun 9 Mar 2014 2:34, edited 1 time in total.
The greater the ignorance, the greater the dogmatism.

Attributed to William Osler, 1902

Martian
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Re: Eva Sapi study about new Borrelia blood culture test

Post by Martian » Sun 9 Mar 2014 1:55

LHCTom,

Thank you for your clear and very informative contributions!


edit to add:
LHCTom wrote:But culturing positive while on Abx for 3 months and just after 7 months did make me wonder.
I can understand that, but I think many people in Lymeland will argue that it just shows how persistent Borrelia burgdorferi is.
Last edited by Martian on Sun 9 Mar 2014 2:17, edited 1 time in total.

nnecker
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Re: Eva Sapi study about new Borrelia blood culture test

Post by nnecker » Sun 9 Mar 2014 2:13

LHCTom,

You seem to be a very smart guy.I am just a lay person.Can you explain why it is so easy for Sapi to culture viable spirokeets after abx treatment and impossible for everyone else,and they even have the advantage of using tissue?

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