Validity of the CDC 2-tiered Test for Late Lyme Borreliosis

Medical topics with questions, information and discussion related to Lyme disease and other tick-borne diseases.
Bagge
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Re: Validity of the CDC 2-tiered Test for Late Lyme Borrelio

Post by Bagge » Mon 29 Apr 2013 14:49

tomgrier wrote:This URL http://www.cdc.gov/lyme/diagnosistreatment/index.html was the connection to the CDC Webinar on Two Tiered Testing from October 2012. It was recently pulled without explanation. In this webinar it was claimed that 2-tiered testing was 97-100 % accurate in Late Stage Neurological Lyme Disease, but no pathology was offered as proof. Throughout the webinar there were many examples of poor scientific method, assumptions, and unsupported statements. Ultimately the conclusion was new and better Lyme testing had to be created, but if the 2-tiered approach is 97-100% accurate why would they need a better test?

Contrary data Testing:
NY Dept Health 1996: found CDC 2-tiered testing missed 82% positive Lyme cases. Dept of Health report to CDC April 15, 1996

Johns Hopkins study 2005: found CDC 2-tiered testing missed 75% of positive Lyme cases IDSA Lyme Disease Guidelines

Commercially available FDA-approved kits are only 36-70% sensitive, : the ELISA assay does not have adequate sensitivity to be part of a two tiered approach to diagnosis.
Johan S. Bakken Journal of Clinical Microbiology, 1997 [35(3): 537-543] IDSA Lyme Disease Guidelines

Six more studies showed 2-tiered testing inadequate and missed on average 56 % of Active Lyme cases.

I might point out that all of this data only pertains to Borrelia burgdorferi, and all the tests use B-31 laboratory strain of Borrelia burgdorferi, but now we have 10 more human pathogenic Lyme strains that the tests cannot address with the same sensitivity as B. burgdorferi.

So how much more false positives would we have seen if the test sera that they used to test the current 2-tiered tests sensitivity was from patients with Borrelia bissetti or Borrelia valasianna or B. americana ????

Tom Grier wrote:It was recently pulled without explanation.
This was the original link: http://www.cdc.gov/lyme/diagnosistreatment/index.html

The original CDC link about Lyme disease testing continues to display a message in large-size type which clearly informs and instructs readers that the "Page Has Moved". It clearly advises the reader to "update your links and bookmarks to the Lyme Disease home page."

This Page Has Moved.

The page you requested has been removed.

Please update your links and bookmarks to the Lyme Disease home page.
The new CDC link about Lyme disease testing is http://www.cdc.gov/lyme/diagnosistesting/index.html
Tom Grier wrote:It was recently pulled without explanation.
It is ridiculous to allege that the video was "pulled without explanation". The video is still there. There is still an updated link displayed on the original webpage re-directing readers to the new CDC Lyme disease page where the video is now located. The CDC sent out a message to all subscribers instructing them that the CDC website about Lyme disease was updated as well. Anyone can subscribe for free. Readers should take advantage of the free subscription rather than depend on misinformation being spread by parties with alternative interests.


http://www.cdc.gov/Other/emailupdates/
The Centers for Disease Control and Prevention (CDC) is happy to offer a free email subscription service, which allows CDC.gov users to receive alerts by e-mail when new information is available. With a subscription profile, you get the updated information on the items of interest to you automatically without having to return to the Web site and check for changes.

This is a free service provided by the CDC. Your email address will only be used to deliver the requested information or to give you access to your user profile.

Subscribe now

<snip>
http://www.lymeneteurope.org/forum/viewtopic.php?f=7&t=4224#p34549
.

Camp Other
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Re: Validity of the CDC 2-tiered Test for Late Lyme Borrelio

Post by Camp Other » Mon 29 Apr 2013 16:33

Regarding that updated link, readers might want to check out the transcript of the video of the latest discussion on Lyme disease diagnosis and testing held at the CDC September 2012:

http://www.cdc.gov/lyme/resources/webin ... script.pdf

Henry
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Re: Validity of the CDC 2-tiered Test for Late Lyme Borrelio

Post by Henry » Tue 30 Apr 2013 20:35

Allow me to try one last time to make my point; forget the mathematical analysis and statistics for the moment.

Because OspC induces an early antibody response, antibody responses to OspC have great significance in the diagnosis of early Lyme disease. However, OspC, the 23 kDa band on an IgG Western blot, is one of the most diverse proteins in the Borrelia proteome; 21 known OspC types have been detected, although 70% of the amino acid sequence is conserved among all 21 known OspC types. Thus, all of these OspC types will be detected as a 23 kDa band on a Western blot used for the diagnosis of Lyme disease since they all contain this conserved sequence; no strain of Borrelia burgdorferi with a different OspC type will be excluded. None of these strains would “fall through the cracks” and escape diagnosis by an IgG Western blot.

Although no single OspC type identifies all seropositive human samples, combinations of as few as two different OspC proteins identified all patients that had anti-OspC antibodies. Although comparable data is not available with respect to the remaining 9 bands that comprise the criteria for an IgG Western blot, keep in mind that the diversity of the proteins that comprise these remaining bands in much less than that of OspC -- the most diverse protein within the Borrelia genome. So, all the concern about whether seronegativity is due to a flawed procedure that is unable to detect diverse strains of Borrelia burgdorferi may really be “much ado about nothing”. One must therefore face the very real possibility that, in the U.S., patients who are seronegative just might not have Lyme disease after all.

See Clinical Immunology 132: 393-400, 2009 for more specific information on this issue.

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LHCTom
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Re: Validity of the CDC 2-tiered Test for Late Lyme Borrelio

Post by LHCTom » Wed 1 May 2013 1:26

Because OspC induces an early antibody response, antibody responses to OspC have great significance in the diagnosis of early Lyme disease. However, OspC, the 23 kDa band on an IgG Western blot, is one of the most diverse proteins in the Borrelia proteome; 21 known OspC types have been detected, although 70% of the amino acid sequence is conserved among all 21 known OspC types. Thus, all of these OspC types will be detected as a 23 kDa band on a Western blot used for the diagnosis of Lyme disease since they all contain this conserved sequence; no strain of Borrelia burgdorferi with a different OspC type will be excluded. None of these strains would “fall through the cracks” and escape diagnosis by an IgG Western blot.

Although no single OspC type identifies all seropositive human samples, combinations of as few as two different OspC proteins identified all patients that had anti-OspC antibodies. Although comparable data is not available with respect to the remaining 9 bands that comprise the criteria for an IgG Western blot, keep in mind that the diversity of the proteins that comprise these remaining bands in much less than that of OspC -- the most diverse protein within the Borrelia genome. So, all the concern about whether seronegativity is due to a flawed procedure that is unable to detect diverse strains of Borrelia burgdorferi may really be “much ado about nothing”. One must therefore face the very real possibility that, in the U.S., patients who are seronegative just might not have Lyme disease after all.
So you are claiming that ALL "real" infected people WILL show positive for the 23kDa band. Then maybe you could can explain why in the recent study done in China,

"A Study of the Technique of Western Blot for Diagnosis of Lyme Disease caused by Borrelia afzelii in China*

http://www.besjournal.com/Articles/Arch ... 811299.pdf

Only found the OspC 23kDa in 12% of the late stage, 5% for NB and only 2% for EM stage

This is not unusual!

If your claim were true, close to 100% would be positive for OspC...

What explains the low percentage of OspC commonly detected as in this study?

Even if your claim were true, the criteria is 5/10 so your argument would need to be true for most if not all 10 chosen antibodies.

What would be your opinion if you tested a patient at one lab and found 3 CDC bands, another lab with another 3 CDC bands but only sharing 41 in common ( so 5/10 detected across 2 tests) and then retested at 3 labs ( national lab, university lab and specialty lab - 2 using Immunetics kit) for C6 and got 3 positives.

Would you claim the C6 is 3 false positives? or

Were the IgG WB at 2 labs both false negatives?

According to the recent CDC Webinar transcript, the only reason the C6 isn't replacing the CDC 2T is their data shows a 1% lower Specificity even though the data on trials varies tremendously.
Utilizing in the third column a C6 approach by itself increased the sensitivity in both Stage 1 and Stage 2 illness, maintained it in Stage 3 illness but realized a slight decrease in specificity of a little bit over 1% which may not seem like a large amount but when you consider the several millions of tests that are performed in the U.S. each year, a 1% increase or decrease in specificity results in many thousands of cases being falsely reported as positive
This is a case of "False Precision Error" where the many systematic errors in the many studies makes it impossible to claim a 1% difference is statistically valid. So the C6 and CDC 2T are essentially equal. So which do you believe? Is the presence of 5 antibodies across 2 WBs meaningful? Or do you blindly follow the criteria or pull your imagination out and dust it off. :woohoo:

So your dusted off imagination wonders if the CDC 2T or C6 is right and you ask some tie breaking questions...


Would you ask if the patient lives in an endemic area? What if it was yes
Would you ask if the patient lives in the country and has routine tick exposure? What if it was yes
Would you ask if the person remembers a bite? What if it was yes and many times over 22 years
What if the patient showed you study done about 1 mile from their home by Dr Lane at UCB and they found the black legged tick nymphs had a >20% infection rate.
What if you did an exhaustive differential diagnosis and found no other explanation for Lyme-like symptoms?

So you are still stuck on the Dogma RIGHT :twisted: .... you send the patient home.... AND YOU WOULD BE WRONG :bonk: Like most imagination-free doctors...

The mess is so obvious...
"Validity of Interpretation Criteria for Standardized Western Blots (Immunoblots) for Serodiagnosis of Lyme Borreliosis Based on Sera Collected throughout Europe"

http://jcm.asm.org/content/37/7/2241.full.pdf+html

"A European Multicenter Study of Immunoblotting in Serodiagnosis of Lyme Borreliosis"

http://jcm.asm.org/content/38/6/2097.full.pdf+html


Doctors would never make it in engineering.... Nothing would ever work.. :ugeek:

Don't bother answering - I know how dogma's work. God told you the CDC is always right so you never question them for fear of going to the wrong place after you die. What if God has a sense of humor and playing a trick on you to test your imagination? :bonk: :bonk: You failed so go there anyway :D
Last edited by LHCTom on Wed 1 May 2013 2:04, edited 1 time in total.
The greater the ignorance, the greater the dogmatism.

Attributed to William Osler, 1902

dlf
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Re: Validity of the CDC 2-tiered Test for Late Lyme Borrelio

Post by dlf » Wed 1 May 2013 1:56

Believe me Henry, I do want to understand and have looked at the article you were referencing.

For anyone looking for access to the article which Henry was using for his reply, it may be found at:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752154/

Henry wrote,Because OspC induces an early antibody response, antibody responses to OspC have great significance in the diagnosis of early Lyme disease. However, OspC, the 23 kDa band on an IgG Western blot, is one of the most diverse proteins in the Borrelia proteome; 21 known OspC types have been detected, although 70% of the amino acid sequence is conserved among all 21 known OspC types. Thus, all of these OspC types will be detected as a 23 kDa band on a Western blot used for the diagnosis of Lyme disease since they all contain this conserved sequence; no strain of Borrelia burgdorferi with a different OspC type will be excluded. None of these strains would “fall through the cracks” and escape diagnosis by an IgG Western blot.
Admittedly, this is somewhat off the topic of this thread which is related to Late Lyme Borreliosis since OspC has most significance with regard to the diagnosis of early Lyme disease and response to OspC diminishes over time. However, I also thought we were discussing the validity of two-tier testing as it is now performed. Not after the testing may be re-formulated. As Henry has pointed out below, "although no single OspC type identifies all seropositive human samples, combinations of as few as two different OspC proteins identified all patients that had anti-OspC antibodies." Since Henry seems to mostly write about the situation in North America and not Europe, I will primarily concentrate my comments to the U.S. testing referenced in the article. In North America two-tier testing is RESTRICTED TO a laboratory grown strain of B31. NOT TWO STRAINS AND NOT THE STRAINS SHOWN TO BE PREFERABLE.

Looking at the article, it is clear from one of the charts:
rA-rU represent purified recombinant OspC proteins; NI, naturally infected serum panels tested positive for B. burgdorferi infection by serological methods. NI P. leucopus, n = 43, is serum panel from naturally infected white-footed mice; NI Dog, n = 38, is serum panel from naturally infected dogs with Lyme disease; NI Human US, n = 25, is serum panel from human North American patients with signs and symptoms of Lyme disease; NI Human EU, n = 40, is serum panel from human European patients with signs and symptoms of Lyme disease; the serum panels included in this analysis tested positive for B. burgdorferi infection by serological methods; shaded gray, OspC proteins that detect the highest titer of antibodies.
For serum panels NI human U.S. tested against type M produced the lowest % positives and K produced the highest at 84%, for European testing the highest % positive was 80% individually for both E and K.

From another chart, showing the various recombinant OspC types with their respective percentage positives and the combinations that produced the strongest reactions:

For the % Positive US LD Panel the combination of B & K produced 96% positives. While individually, type A was 68%, type B was 72%, E & F both each 80%, type I was 76% K was 84% and L was 68%. I repeat A was 68% - because A is the OspC type found in the commercially available North American tests.

In Europe E combined with F or K produced 100% and individually the types ranged from A at 74% to E or K at 91%
From the article:
Although it has been determined that the polymorphism of OspC is due to positive selection favoring diversity at the amino acid level in the variable region [37] and that the immunodominant epitopes of OspC reside in the variable domains of the protein [29] it would appear that common epitopes present in OspC types B, E, F and K detect most anti-OspC antibodies present in serum samples from seropositive patients infected with B. burgdorferi. Contrary to the dogma, our results indicate that OspC proteins belonging to these four genotypes may be among the best candidates to develop additional diagnostic tools for early Lyme disease. As with all serodiagnostic assays, caution should be used given that up to 13% of samples, with proven anti-B. burgdorferi antibodies from three different hosts, did not react to any of the 16 OspC tested. This highlights a source of false-negative results that could indirectly lead to the increase in the incidence of late Lyme disease.

In all four serum panels we observed that a number of individuals reacted to all 16 OspC types and that a number of samples did not have antibodies to any OspC

Our conclusions are also supported by the observation that most patients infected with B. burgdorferi (regardless of strain type) develop anti-OspC antibodies that bind to OspC belonging to genotype A used in commercial serodiagnostic assays in both ELISA and immunoblot formats.
WHERE IN THE MATERIAL IN THIS ARTICLE, IS IT SHOWN THAT THERE IS PROOF TO SUPPORT THIS CONCLUSION MADE IN THE LAST SENTENCE? Bearing in mind that OspC A only reacted positively to 68% of the serum panels. Is 68% the same as "most"?
Our results suggest major cross-reactivity between OspC antibodies. Although the protective OspC epitopes are genotype-specific, shared OspC epitopes elicit detectable antibody responses for use in diagnostic applications.
Again, where is the proof of this shown in the articlewith regards to B31 - typeA OspC ? Is suggestion the same as proof?
However, the best combination of rOspC proteins for LD diagnosis differed on the two continents. rOspC types B and K identified 96% of the US LD patients, with one patient’s serum reacting only to type J. Over 76% of North American patients reacted positively with type J, suggesting that a diagnostic assay based in these three proteins may decrease false negative results.
So this may pave the way for a new testing protocol, but what does it show about the validity of the two-tier tests that have been done for years and are still in use?

Henry wrote,
Although no single OspC type identifies all seropositive human samples, combinations of as few as two different OspC proteins identified all patients that had anti-OspC antibodies. Although comparable data is not available with respect to the remaining 9 bands that comprise the criteria for an IgG Western blot, keep in mind that the diversity of the proteins that comprise these remaining bands in much less than that of OspC -- the most diverse protein within the Borrelia genome. So, all the concern about whether seronegativity is due to a flawed procedure that is unable to detect diverse strains of Borrelia burgdorferi may really be “much ado about nothing”. One must therefore face the very real possibility that, in the U.S., patients who are seronegative just might not have Lyme disease after all.
Seronegativity is a factor of both of the two-tier tests, not just the Western Blots. I am one of many who was non-reactive to the B31 Elisa, the only test available in Ontario when I was finally tested almost two years after my symptoms onset and when I finally managed to get my physician to ask an Infectious Diseases doctor how to order Lyme serology. I could not have a Western blot done here. At the same time as that test was done I had high white blood cell counts above reference range, similarly high lymphocyte counts and I had hypogammaglobulinema. Four months prior, I was tested by Igenex and was IgG positive but CDC negative with 4 of the possible 10 bands being positive and one other of the 10 bands indeterminate, plus I was positive for 31kDa. On top of that, at the time I tried to get testing for the C6 Elisa and European Lyme I was unable to get those tests done due to lack of knowledge on my physician's part and the Infectious Diseases doctor he consulted, despite his being told that I had exposures in North America and two areas in Europe, (one known for Borrelia garinii and the other which was later shown to be endemic for Borrelia lusitaniae). So maybe I don't have Lyme, although other physical findings, tick exposures and response to treatment point in that direction.

If you were in my shoes, wouldn't you be concerned, Henry?

Lorima
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Re: Validity of the CDC 2-tiered Test for Late Lyme Borrelio

Post by Lorima » Fri 3 May 2013 14:45

The original post in this thread, from March 19, 2013: 
TicksSuck wrote:I have asked a seemingly knowledgeable person before in other topics, but never got a specific answer... Maybe there was too much noise from shillers and the question got lost, or (what I suspect), the person just don't have an answer. I will therefore ask more generally in a new noise-free topic.

Can somebody please provide me with a study that shows that the 2-tired test advocated by the CDC for Lyme Disease (http://www.cdc.gov/lyme/healthcare/clin ... otier.html) is sensitive in cases of late stage (>30 days) disease, but doesn't suffer from selection bias?
It seems that the answer is, no. 
"I have to understand the world, you see."
Richard Feynman

TicksSuck
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Re: Validity of the CDC 2-tiered Test for Late Lyme Borrelio

Post by TicksSuck » Mon 6 May 2013 14:48

In another thread, Henry said:
Incidentally, your argument for circular reasoning is flawed. Many studies used to provide information on the sensitivity and validity of 2-tiered testing were based on panels of well-characterized specimens obtained at later times from patients who were culture positive, and/or had EM rashes. It is here that convalescent serum, as well as sera acquired late during infection; obviously, such sera would be expected to be seropositive. In other words, specimens were used in which there was little doubt that they came from patients who have/had Lyme disease. To evaluate the limitations of a diagnostic test based on the use of such specimens makes good sense to me. Furthermore, different panels of such specimens were used in the different studies described, all of which gave the same results. So, is everyone involved in such work stupid and using circular reasoning to make their point? I rather doubt that very much. Too many people of integrity are involved to allow for such a thing. And please spare us the quotes from your selective readings.......

Henry
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Re: Validity of the CDC 2-tiered Test for Late Lyme Borrelio

Post by Henry » Mon 6 May 2013 19:53

The answer is YES. See Table 3 in : http://cid.oxfordjournals.org/content/4 ... l.pdf+html. Also, examine the frequency of bands for the IgG Western blot (Table 2). The data are very convincing and consistent with the CDC criteria for a positive Western IgG blot..

TicksSuck
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Re: Validity of the CDC 2-tiered Test for Late Lyme Borrelio

Post by TicksSuck » Mon 6 May 2013 21:33

Henry wrote:The answer is YES. See Table 3 in : http://cid.oxfordjournals.org/content/4 ... l.pdf+html. Also, examine the frequency of bands for the IgG Western blot (Table 2). The data are very convincing and consistent with the CDC criteria for a positive Western IgG blot..
Hmmmm, no. The answer is still no:

From the study you link:
Steere AC, McHugh G, Damle N, Sikand VK. Prospective study of serologic tests
for lyme disease
. Clin Infect Dis. 2008 Jul 15;47(2):188-95. doi: 10.1086/589242.
PubMed PMID: 18532885
Inclusion criteria for patients with later manifestations of
the infection.


Neuroborreliosis was defined clinically as meningitis,
cranial neuropathy, peripheral neuropathy, or radiculoneuropathy.
Except for patients with cranial neuropathy, these
patients were required to have CSF pleocytosis or electromyographic
evidence of an axonal polyneuropathy [8]. Cardiac
involvement was defined by the presence of acute atrioventricular
nodal block. Lyme arthritis was defined as inflammatory
arthritis in 1 large joint. In all patients with neurologic,
cardiac, or joint involvement, a serologic result positive for B.
burgdorferi by ELISA and Western blot was required for case
inclusion [5]
.
The question was:
Can somebody please provide me with a study that shows that the 2-tired test advocated by the CDC for Lyme Disease (http://www.cdc.gov/lyme/healthcare/clin ... otier.html) is sensitive in cases of late stage (>30 days) disease, but doesn't suffer from selection bias
If there are many studies as you say that were based on panels of well-characterized specimens obtained at later times from patients who were culture positive, and/or had EM rashes, what are they? I'm interested in the details of these studies.

dlf
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Re: Validity of the CDC 2-tiered Test for Late Lyme Borrelio

Post by dlf » Mon 6 May 2013 22:10

Really Henry........ once again I think you are reading the article you are referencing and NOT noting a key sentence or two. As regards late lyme neuroborreliosis and arthritis selection bias was an issue for this study.
Inclusion criteria for patients with later manifestations of
the infection. Neuroborreliosis was defined clinically as meningitis,
cranial neuropathy, peripheral neuropathy, or radiculoneuropathy.
Except for patients with cranial neuropathy, these
patients were required to have CSF pleocytosis or electromyographic
evidence of an axonal polyneuropathy [8]. Cardiac
involvement was defined by the presence of acute atrioventricular
nodal block. Lyme arthritis was defined as inflammatory
arthritis in 1 large joint. In all patients with neurologic,
cardiac, or joint involvement, a serologic result positive for B.
burgdorferi by ELISA and Western blot was required for case
inclusion [5].
If a serologic result positive for B. burgdorferi by ELISA and Western blot was REQUIRED for case inclusion for these patients.......how could any of them have tested negative to IgG Western blot? The chart is meaningless with regards to these late stage patients and once again demonstrates circular reasoning. Why take patients who are required to test positive to two-tier testing and then use the results to "prove" that two-tier testing is 100% sensitive? What this has established, (or at the very least suggests), is that there might be a serious problem with peer-review.
Serologic methods. Serologic results were determined by 2-
tier sonicate ELISA and Western blot and by VlsE C6 peptide
ELISA, as described elsewhere [6, 10–12]. Both ELISAs were
noncommercial, in-house tests. For the sonicate ELISA, the
antigen preparation was derived from B. burgdorferi strain G39/
40
[10].
I am also wondering, since we are discussing two-tier testing as it is currently formulated, why they decided in this study to use an ELISA with strain G39/40 INSTEAD of the commercial laboratory B-31 ELISA that the North American public was tested with up until very recently. Is it possible that the commercial ELISA would have been negative for too many of the well-characterized CDC serum samples, which after-all were collected from localized areas.......so many years ago? Hopefully the new CDC serum sample bank will be representative and reflective of more Borrelia strains and combinations of strains which are now appearing in a single tick and which presumably are also infecting patients with multiple strains. Infections in the natural wild type ticks have evolved since the original samples were collected and much later used for your quoted study.

As I was about to post this I noticed TicksSuck has posted a very similar reply to mine. But, I do also want to say that some of the very early studies done, when the serology tests were being developed were in fact based on positive culture and that really was the "Gold Standard" for determining sensitivity. That's one reason that the reported sensitivity was generally lower than what the IDSA/CDC are claiming now. It will take me a while to find the references. But there are also other references available that show that serology is not particularly sensitive when compared to patients who were characterized by culture and PCR. I have a few tucked away and all of them might not be applicable but here is a quick list with out double checking exactly which aspect of this issue is being addressed. Also, sorry I don't have time to post the web links but if you look for them on the net, all of them were free full text articles.


1. Aguero-Rosenfeld ME, et al, Serodiagnosis in early Lyme disease, J. Clin. Micro 1993:31 (12): 3090-3095;

2. Impact of Clinical Variables on Borrelia burgdorferi Specific Seropositivity on Acute-Phase Sera from Patients in North America with Culture confirmed Early Lyme Disease, Gary P. Wormser, et al, Aug. 20, 2008, PMC:2565926

3. Bratisl Lek Listy 2010; 111(3): 153-155 PMID: 20437826. This study is titled, "Our experience with examination of antibodies against antigens of Borrelia burgdorferi in patients with suspected Lyme disease", by Durovska J, Bazovska S, Ondrisova M, Pancak J.

The results read: "All patients," (in the study N 32)," had specific anti-borrelial antibodies confirmed by using western blot in spite of negative Elisa. Immunological investigations revealed a deficiency of cellular immunity in all patients and in a part of them (15.6%) a deficiency of humoral immunity was also found.

4. Oksi J., et al, Antibodies against whole sonicated Borrelia burgdorferi spirochetes, 41-kilodalton flagellin & P39 protein in patients with PCR or culture proven late lyme borreliosis. J Clin Microbiol 1995 Sept; 33(9) 2260-4.

5. Chinielewski T., et al, Improvement in the laboratory recognition of Lyme borrelliosis with the combination of culture and PCR methods. Mol Diag 2003; 7 (3-4); 155-162.

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