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

Post by radicale » Thu 25 Apr 2013 1:44

Henry wrote:Radicale: Sorry, my mistake. I acknowledge your point with respect to European borreliosis where there are at least 3 genera involved and much more strain diversity than in the U.S. upon which my comments were based. I think I have said all that I have to say on this topic.
Strain diversity in the USA matters as well.

Effect of Borrelia burgdorferi genotype on the sensitivity of C6 and 2-tier testing in North American patients with culture-confirmed Lyme disease.
Wormser GP, Liveris D, Hanincová K, Brisson D, Ludin S, Stracuzzi VJ, Embers ME, Philipp MT, Levin A, Aguero-Rosenfeld M, Schwartz I.
Source
Division of Infectious Diseases, Department of Medicine, New York Medical College, Valhalla, NY 10595, USA. gary_wormser@nymc.edu

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.
METHODS:
C6 testing and 2-tier testing were performed on acute-phase serum samples obtained from >158 patients with erythema migrans for whom the genotype of the borrelial isolate was defined on the basis of an analysis of the 16S-23S ribosomal DNA spacer region and/or on the genetic variation of the outer surface protein C gene (ospC). The sonicated whole cell-based enzyme-linked immunosorbent assay, the immunoblots used in the 2-tier testing, and the C6 assay all used antigens from B. burgdorferi sensu stricto strain B31.
RESULTS:
The sensitivity of C6 testing (69.5%) was greater than that of 2-tier testing (38.9%) (P<.001); the difference in sensitivity, however, was statistically significant only for patients infected with 2 of the 3 ribosomal spacer type-defined genotypes. The lower sensitivity of 2-tier testing was attributable to the low sensitivity of the immunoblot tests, rather than the first-tier enzyme-linked immunosorbent assay. 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 (P=.07); this relationship was not observed with C6 testing.
CONCLUSIONS:
Lack of sensitivity of the C6 test because of strain diversity seems less likely to be a limitation of this serologic test, compared with 2-tier testing in North American patients with early Lyme disease.
If you take the time to read through it you will see that the 2-tier test has a sensitivity ranging from 0-53%% for early (culture-positive) Lyme disease, highly dependant on the strain.

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

Post by Camp Other » Thu 25 Apr 2013 2:53

That paper got me thinking about something. It states:
The sensitivity of 2-tier testing varied according to RST genotype (P < .02), with RST1-infected patients more than twice as likely as RST3-infected patients to be seropositive (54.3% vs. 25.0%, P = .008).
Has anyone demonstrated evidence there is a connection between patients who were infected with RST3 and persisting symptoms?

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

Post by LHCTom » Sun 28 Apr 2013 6:39

One can easily see why the CDC 2T is flawed by looking at the raw data in the latest WB study in China. Even though the study is for B. afzelii in China, there is no fundamental reason why Borrelia Burgdorferi in the US would vary that much since the immune response is similar and surface proteins are similar. One would not expect a sufficient difference to support a CDC 5/10 IgG criteria plus the choice of the the actual 10 antibodies is dubious at best.

One wildcard is if this study or the US studies got the antigens used in the WB right for the genotype surface antigen variants. Nobody seems to account for the variation due to even strain given they swear only Bb infects people in the US. Gary Wormser mentions this problem but always just uses good old B31 for the antigens. The Chinese used one sample they cultured for their antigens. Maybe everyone gets it wrong for this reason. Every study uses people infected with a variety of strains. Every study uses one or maybe 2 strains/antigens for their WB. If the precise variation and whether a few or many of the actual infection strains have surface antigens with sufficiently different epitopes, they may not show up on the WB having not been transferred.

A moment on sensitivity

True positive = correctly identified
False positive = incorrectly identified
True negative = correctly rejected
False negative = incorrectly rejected

sensitivity = number of true positives/ ( number of true positives + number of false negatives)

So its critical one must know with near certainty, the number of true positives ( this is where many studies fail )

Its also critical knowing the False negatives = incorrectly rejected

Its also extremely critical the study population contain a reasonable mix of infection strains/species that reasonably matches that which will be encountered in the clinical setting. A study with only patients form the Northeast does not correctly represent the US diversity. For example, failing to use strains found in CA or the South will cause inaccurate results since the CDC 2T is sensitive to strain and species diversity - Gary Wormser.

Knowing the number of true positives requires a test such as a culture or PCR that is independent of the test under study i.e. CDC 2T. That test must be very good. The new ALS culture may be good enough especially when combined with amplified high volume blood PCR. These two get an accurate mix of stains and number of true positives. :woohoo:

Now one must know the False negatives = incorrectly rejected. If one knows the REAL number of true positives and the mix of strains matches the clinical setting, then negatives on the CDC 2T that are known to be true positives, provides all the data needed. :D

Leaving off high cross reacting antibodies gets the best specificity while using the most commonly ocurring antibodies helps yield the best sensitivity/specificity - tradeoff

Back to the Chinese study example: :bonk:


So what does the raw data show? It shows that the US IgG 5/10 requirement for positive is far too biased toward specificity versus sensitivity. No wonder it fails so often. The US researchers routinely claim that virtually 100% of late NB is CDC positive. That is also absurd based on this data. :?:

The ALS culture is undergoing validation at 2 independent labs. If it performs as advertised, then we can finally know what species and strains are in these study participants. Then it will be possible to properly "tune" the antigens used in the WB to be sure strain diversity ( and sometimes species) isn't screwing the CDC 2T test up. I suspect strain diversity, a test good enough for knowing true positives and study selection bias are the culprits. :D
"Study of the Technique of Western Blot for Diagnosis of Lyme Disease caused by Borrelia afzelii in China"

http://www.besjournal.com/Articles/Arch ... 77498.html
They show in table 1 the percentage of each category with a positive antibody for EM NB Late plus controls and for IgM

Lets focus on the IgG

Table 1 shortened:

AB Percentage
kDa EM NB late

P83/100 6 11 18
P75 3 2 7
P66 3 2 6
P58 5 5 11
P43 2 2 5
P41 15 20 25
P39 5 13 10
OspA 35 2 5 9
OspA 32 12 12 17
P30 4 3 4
P28 2 2 7
OspC 22 2 5 12
P17 3 7 7
P14 8 3 18

It doesn't take a ROC analysis to see that P83/100 and P14 which are the most common are only 18% of the late category population each. There is a 4/5 probability one will not have each of these most common antibodies. The odds only get worse with the remaining antibodies. For simplicities sake, lets assume the 10 antibodies chosen in the ROC analysis all had an occurrence percentage of 20%. That's better than the 2 best P83/100 and P14 and far better than P43 and P30 at 5% and 4% respectively. But lets take this optimistic and easy to see case of them all being 20%. The odds of having any one is 1/5. If there are 10 chosen than 1/5 x 10 = 10/5 = 2. So in this very optimistic case where they all appear at 20% ( but they don't), then the average test would have 2/10. :?:

All the antibodies together only add up to 157%. That means the average study participant only had 1.57 antibodies. In other words, the odds of having one antibody is excellent while the odds of 2 is poor. So they then proceed to do the ROC analysis to see what x is where 1/x is the criteria as a specificity tradeoff plus the best set. One can see by inspection that the the ratio for IgG must have a 1 in its numerator or the sensitivity will plummet. That is the CDC 2T fatal flaw.

The actual 10 antibodies chosen are 83/100, 58, 39, OspB, OspA, 30, 28, OspC, 17 and 14. There total occurrence rate is 113%. That means the odds of having one antibody in the 10 is 113% or good. So 1/10 makes sense. The choice of actual antibodies comes from the ROC analysis which is used to get the best sensitivity versus specificity tradeoff. 41 is eliminated because even though it helps sensitivity, its disastrous to specificity due to the high cross reactivity. Its removed form the IgG criteria due to its lowering of specificity more than its sensitivity gain. The US boys blew this one. :bonk:

The ROC analysis is used to choose the best sensitivity versus specificity tradeoff by examining sets of antibodies using the most common ones first since that simplifies the analysis and is obvious. They came up with 1/10 for IgG and their 10 antibodies are not the same as in the US. I would expect the B Burgdorferi data in the US to differ possibly in preferred set but not significantly in frequency of occurrence if the selection bias used by most US researchers to be corrected. Looks like the Chinese are smarter than US researchers. That's scary since they are slowly cleaning our clock elsewhere. :twisted:

They use the ROC analysis to chose which antibodies and the value for the denominator. Recall the US criteria is 5 = numerator and 10 is the denominator. Both the choice of antibodies and the 5/10 is absurd if the US data for LB is anything like this data. :?:

Yes this is not B Burgdorferi :!: but the immune response and surface proteins are sufficiently similar that one would expect roughly similar results. In order for the results to support 5/10 for IgG, the sum of the late NB percentages used for the denominator set would need to add up to over 500%. That would mean on average, 5 antibodies are seen. This data clearly shows only 1 as a valid numerator. :D :D :D

The total EM column doesn't even add up to 100%. That suggests even their criteria of 1/10 from P83/100, P58, P39, OspB, OspA, P30, P28, OspC, P17, and P14 will have poor sensitivity. :cry:

Results Criteria for a positive diagnosis of Lyme disease were established as at least one band of P83/100, P58, P39, OspB, OspA, P30, P28, OspC, P17, and P14 in the IgG test

and at least one band of P83/100, P58, P39, OspA, P30, P28, OspC, P17, and P41 in the IgM test. For IgG criteria, the sensitivity, specificity and Youden index were 69.8%, 98.3%, and 0.681, respectively; for IgM criteria, the sensitivity, specificity and Youden index were 47%, 94.2%, and 0.412, respectively.
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Henry
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Re: Validity of the CDC 2-tiered Test for Late Lyme Borrelio

Post by Henry » Sun 28 Apr 2013 14:56

What must be kept in mind is that these Western blot bands represent an array of epitopes, SOME of which might be strain specific whereas several others many be shared by ALL strains of Borrelia burgdorferi. During the past several years, many different clinical isolates of B. burgdorferi have been obtained and characterized, all of which were derived from patients who were diagnosed for Lyme disease by means of the two-tiered test. These clinical isolates are not so different from each other that one needs to develop a unique laboratory test for their detection. None of them would have "fallen through the cracks", thereby avoiding diagnosis. The same can be said for the diagnosis of other infectious disease. The basis for many of the laboratory tests used in the U.S. is the presence of shared invariable epitopes among all strains of Borrelia burgdorferi, although adjustments certainly must be made in Europe to detect infections caused by other species of Borrelia; this is in fact done for the Western blots that they use in Europe for diagnosis.

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

Post by LHCTom » Sun 28 Apr 2013 20:33

What must be kept in mind is that these Western blot bands represent an array of epitopes, SOME of which might be strain specific whereas several others many be shared by ALL strains of Borrelia burgdorferi.
"Some of which may be strain specific" - This is exactly why using a high numerator in the criteria fails!

During the past several years, many different clinical isolates of B. burgdorferi have been obtained and characterized, all of which were derived from patients who were diagnosed for Lyme disease by means of the two-tiered test.


If one is trying to use logic to validate assumptions about the CDC 2T test, then it fails when the CDC 2T test is used to select the cases that support the argument. If these isolates where found using the CDC 2T test, then the epitopes on these isolates are of course detectable by the CDC 2T test. What about the set of isolates rejected by the CDC 2T test. Its not valid to use a test to validate itself. This is the type of "poor reasoning" that has created the problem.
These clinical isolates are not so different from each other that one needs to develop a unique laboratory test for their detection. None of them would have "fallen through the cracks", thereby avoiding diagnosis.
Again, these isolates where found using the test being validated. The boundary where a strain is differentiated from a species is becoming more and more fuzzy based on the latest molecular techniques. As in Europe, a different antigen set and criteria was used due to diversity. Even Gary Wormser admits the CDC 2T test and C6 test are both strain and species sensitive. Many cases are falling through the cracks but its the rigid 5/10 criteria dominating rather than epitope variations but they do contribute.

The same can be said for the diagnosis of other infectious disease.
Borrelia is among the most antigenic of infectious microbes. The performance of an antibody based test is well known to be impacted by highly antigenic microbes since they keep altering their surface protein epitopes. This causes the antibodies to fail to recognize the new epitopes.
The basis for many of the laboratory tests used in the U.S. is the presence of shared invariable epitopes among all strains of Borrelia burgdorferi, although adjustments certainly must be made in Europe to detect infections caused by other species of Borrelia; this is in fact done for the Western blots that they use in Europe for diagnosis.
Yes there are shared epitopes that don't change. But when a test requires 5 out of a set of 10, then it becomes very sensitive to the ones that do change with genotype evolution. This is why most countries require 1/10 or 2/10 as the criteria ratio. They understand this sensitivity and have properly analyzed the sensitivity versus specificity tradeoff. Maybe our researchers need to go back and crack open their Probability and Statistics textbooks or get help from a real mathematician - or someone with common sense would be helpful. :woohoo:
The greater the ignorance, the greater the dogmatism.

Attributed to William Osler, 1902

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

Post by Henry » Mon 29 Apr 2013 2:36

The number of bands selected was determine by a very careful statistical analysis of the type and numbers of bands that appear when well-characterized specimens were assayed. They include specimens from patients with early acute Lyme disease, convalescent sera, serum from Lyme disease patients with arthritis and/or neurological complications, late Lyme disease, as well as meaningful controls that included normal serum from patients living in endemic and/or non -endemic areas, and patients with other types of infections (e.g., syphilis). The selection of 5 of 10 bands for the IgG Western blot was based on the lowest number of bands showing the highest reactivity for specimens from patients diagnosed as having Lyme disease, but no reactivity in the control specimens. Hundreds of well-characterized specimens were used in the analysis by people who know more about statistics and their proper application in such studies. THey know what they are doing.

Furthermore, although the 23 kDa band (OspC) shows as a single band, it is composed of dozens ( if not more) of different epitopes that vary from strain to strain. Be that as it may, the band has diagnostic value simply because all strains of Borrelia burgdorferi have and OspC ( 23 kDa) band), although the epitopes within that band may not be identical between clinical isolates. The same can be said of the other 10 bands that are relevant.

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

Post by dlf » Mon 29 Apr 2013 3:43

Henry wrote,"These clinical isolates are not so different from each other that one needs to develop a unique laboratory test for their detection. None of them would have "fallen through the cracks", thereby avoiding diagnosis."

LCHTom wrote, "Again, these isolates where found using the test being validated. The boundary where a strain is differentiated from a species is becoming more and more fuzzy based on the latest molecular techniques. As in Europe, a different antigen set and criteria was used due to diversity. Even Gary Wormser admits the CDC 2T test and C6 test are both strain and species sensitive. Many cases are falling through the cracks but its the rigid 5/10 criteria dominating rather than epitope variations but they do contribute."
I would like to look at some of the specifics in this debate. Many people fall through the cracks when they are non-reactive to the first tier test. This can happen for a variety of reasons, among them strain diversity, genotype variation, individual immune system variability, the level set for reactivity being based on people with very strong immune reactions, etc. It appears that in many places around the world that the C6 Elisa is starting to replace whole cell Elisa testing. In theory this would appear to make sense, in that the C6 measures an invariable region of VlsE. In practice, it would seem there is more diversity than is generally being accounted for:
Epitope Length, Genospecies Dependency, and Serum Panel Effect in the IR6 Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Borrelia burgdorferi▿
Maria J. C. Gomes-Solecki 1 ,3 ,
Luciana Meirelles 1 ,
John Glass 2 and
Raymond J. Dattwyler 1 ,3 ,*
- Author Affiliations

1New York Medical College, Valhalla, New York 10595

2Brookhaven National Laboratory, Upton, New York 11973

3Biopeptides Corp., Valhalla, New York 1059

http://cvi.asm.org/content/14/7/875.full

ABSTRACT
In the absence of erythema migrans, the basis for diagnosis of Lyme disease is the demonstration of an antibody response against Borrelia burgdorferi in an appropriate clinical setting. The C6 enzyme-linked immunosorbent assay, based on the IR6 region of VlsE, has become widely used in both the United States and Europe. We mapped the antigenic epitopes of IR6 to a shorter sequence that is equivalent in sensitivity and specificity to the full-length IR6 25-residue peptide. In addition, we observed significant differences in sensitivity between serum panels (60 to 100%), indicating that the selection of the serum panels can shape the apparent overall sensitivity of the assay. Contrary to prior reports, the assay sensitivity is greater when the IR6 peptide is derived from the sequence of the same infecting Borrelia genospecies. Using our North American panels and the two panels obtained from European Lyme disease patients, we determined that the IR6 assay that is based on a single genospecies of Borrelia spp. is not optimal for use as a universal diagnostic assay for Lyme disease.
Since North American testing has so far been mostly based on laboratory grown B-31, it has limitations. But what about some other strains and the apparent reactivity to the C6 Elisa?
Let's look at another strain of Borrelia burgdorferi which is found in North America,
Strain N40.
From:
Lymphoadenopathy during Lyme Borreliosis Is Caused by Spirochete Migration-Induced Specific B Cell Activation
Stefan S. Tunev,1,2,¤ Christine J. Hastey,1,4 Emir Hodzic,1 Sunlian Feng,1 Stephen W. Barthold,1,2,3,4 and Nicole Baumgarth1,2,3,4,*
Jenifer Coburn, Editor

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3102705/

<snip>
We did not include the VlsE protein in our studies, a surface-protein thought to subvert the immune response to B. burgdorferi through extensive genetic variation within the host. However, the N40 strain of B. burgdorferi, which we have used here, does not seem to express this protein, based on transcriptional analysis of the IR6 region of vlsE. Moreover, we found no evidence of seroconversion to the C6 antigen of vlsE from strain B31 (S. W. Barthold, unpublished). Recent sequence analysis of the N40 genome has confirmed that N40 vlsE and BBK01 are on different plasmids and that the vlsE locus is indeed significantly different compared to B31, the commonly used VlsE-expressing Borrelia-strain.
<snip>
OK, so you happen to have the bad luck of being infected with strain N-40, you are unlikely to react to the C6 Elisa.
To further compound the problem with N-40:
See

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3318418/
Infect Immun. 2012 Apr;80(4):1519-29. Epub 2012 Jan 30.

Detection of established virulence genes and plasmids to differentiate Borrelia burgdorferi strains.

Chan K, Casjens S, Parveen N.

Source

Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey, USA.

Abstract

Borrelia burgdorferi sensu stricto is the major causative agent of Lyme disease in the United States, while B. garinii and B. afzelii are more prevalent in Europe. The highly complex genome of B. burgdorferi is comprised of a linear chromosome and a large number of variably sized linear and circular plasmids. Many plasmids of this spirochete are unstable during its culture in vitro. Given that many of the B. burgdorferi virulence factors identified to date are plasmid encoded, spirochetal plasmid content determination is essential for genetic analysis of Lyme pathogenesis. Although PCR-based assays facilitate plasmid profiling of sequenced B. burgdorferi strains, a rapid genetic content determination strategy for nonsequenced strains has not yet been described. In this study, we combined pulsed-field gel electrophoresis (PFGE) and Southern hybridization for detection of genes encoding known virulence factors, ribosomal RNA gene spacer restriction fragment length polymorphism types (RSTs), ospC group determination, and sequencing of the variable dbpA and ospC genes. We show that two strains isolated from the same tick and both originally named N40 are in fact very distinct. Furthermore, we failed to detect bbk32, which encodes a fibronectin-binding adhesin, in one "N40" strain. Thus, two distinct strains that show different plasmid profiles, as determined by PFGE and PCR, were isolated from the same tick and vary in their ospC and dbpA sequences. However, both belong to group RST3B.
So much for N-40. How about strain 2591?
Comparative evaluation of three different ELISA methods for the diagnosis of early culture-confirmed Lyme disease in Italy
Antonella Marangoni 1 ,
Monica Sparacino 1 ,
Francesca Cavrini 2 ,
Elisa Storni 2 ,
Valeria Mondardini 3 ,
Vittorio Sambri 1 and
Roberto Cevenini 1

http://jmm.sgmjournals.org/content/54/4/361.full

BB ss Strain 2591 chart showing discrepancies between C6 Elisa and Ecoblot Western Blots which are very specific to this strain. In this chart look at the negative C6 Elisa reporting shown with positive Ecoblot Western Blots IgG and IgM.

http://jmm.sgmjournals.org/content/54/4 ... nsion.html

So, if you are operating under two-tier testing with the C6 Elisa and happen to be infected with Strain 2591, chances are it won't show up as reactive on the Elisa and consequently you won't be tested with the Western Blots.

This study was done in Italy, but that doesn't mean that strain 2591 is specific to Europe. In fact, here is an excerpt from a U.S. patent registration referring to the same strain found in Connecticut.


http://www.patentgenius.com/patent/5620862.html

Inventor: Padula Date Issued: April 15, 1997 Application: 08/158,353 Filed: November 24, 1993
SUMMARY OF THE INVENTION

This invention is based upon the discovery that sera from patients with early Lyme disease contain predominant IgM reactivity to a major 23 kDa protein (p23; also referred to herein as OspC) from Borrelia burgdorferi strain 2591, an isolate foundin Connecticut. This strain was found to abundantly produce OspC. The p23 gene from strain 2591 was cloned and sequenced. The protein deduced therefrom contained 212 amino acids and had a molecular weight of 22,250 kDa. Purified and recombinant formsof OspC protein were produced as a target antigen and tested in Enzyme Linked Immunosorbant Assay (ELISA) and Western Blot assays.

Detection of B. burgdorferi-specific IgM antibodies in 74 individuals with culture positive erythema migrans and 76 controls without Lyme disease were determined using a whole cell (WC) ELISA, immunoblot and recombinant OspC (rOspC) ELISA. Withall test results there was a statistically significant association between the duration of disease and the frequency of a positive result. The rOspC ELISA had a positive predictive value of 100% and a negative predictive value of 74%. Simlar resultswere obtained with the whole cell ELISA and immunoblot.

Based upon these results, recombinant p23 can be used in diagnostic assays (serological and cellular) to detect early stages of Lyme disease. Methods for detecting early Lyme disease, diagnostic reagents therefor, kits containing the reagents,and method for preventing Lyme borreliosis are described. The invention provides the advantages of immunoassay standardization due to use of recombinant forms of OspC; early detection of the humoral response in infected individuals; and antigenuniformity due to phenotype stability of strain 2591. The rOspC ELISA is equally or more sensitive and specific than currently used diagnostic tests for early Lyme disease.


The invention is based upon the discovery that Borrelia burgdorferi strain 2591, a Connecticut isolate, abundantly expresses OspC. This is unique in contrast to most North American strains of B. burgdorferi which do not express the OspC protein. For example, strain B31 is commonly used as a serologic assays for Lyme disease. OspC is an immunodominant antigen as it elicits a humoral antibody response in mammals with early Lyme disease. Thus, purified and recombinant forms of the OspC proteincan be used as a diagnostic tool for screening/detecting early Lyme disease.
Analysis of the strain 2591 sonicate by SDS-PAGE stained with Coomassie Blue demonstrated the expression of an abundant protein (p23) with a mobility corresponding to a molecular weight of approximately 23 kDa. Examination of the protein patternfrom B. burgdorferi strain B31 isolated in North America demonstrated the lack of a detectable protein of 23 kDa. Both strains had been grown under the exact same conditions. An immunoblot of these electrophoretic patterns with serum from a patientwith early Lyme disease demonstrated strong IgM reactivity to the 41 kDa flagellar antigen and relatively weak reactivity to 37, 39 and 75 kDa antigens from both strains. However, only with the strain 2591 sonicate was there detectable IgM reactivitywith the 23 kDa protein. No additional antigen reactivity was detected in the lane containing the sonicate of strain B31.
I included this as also being pertinent to the next quote, since B31 would appear to not express 23 kDa, one of the 5 of 10 bands used as the criteria for CDC positive reactivity for Western blots. So, for strain B31 it seems that the real criteria is 5 out of 9 bands, (or possibly less, more research would be required to find out exactly how many).

Henry wrote, "The basis for many of the laboratory tests used in the U.S. is the presence of shared invariable epitopes among all strains of Borrelia burgdorferi, although adjustments certainly must be made in Europe to detect infections caused by other species of Borrelia; this is in fact done for the Western blots that they use in Europe for diagnosis."

LHCTom wrote, "Yes there are shared epitopes that don't change. But when a test requires 5 out of a set of 10, then it becomes very sensitive to the ones that do change with genotype evolution. This is why most countries require 1/10 or 2/10 as the criteria ratio. They understand this sensitivity and have properly analyzed the sensitivity versus specificity tradeoff. Maybe our researchers need to go back and crack open their Probability and Statistics textbooks or get help from a real mathematician - or someone with common sense would be helpful."
Is there a mathematician on this forum who would care to work out the odds or probabilities based on 5 out of 9 possible bands for any given strain versus the 10 that the CDC is implying should be possible for all strains?

Somewhat off topic.....does anyone think that the semantics of what constitutes "Lyme" in North America being a disease solely based on Borrelia burgdorferi senso stricto begin to address the issues of a patient with non-specific symptoms who happens to live in an endemic area but has also travelled to any number of places in the world where other Borrelia species are common? How many physicians know enough to test for TBRF Borrelia, including several found in North America before sending their patient home with no answer except for, "Well, you don't have Lyme."?

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

Post by tomgrier » Mon 29 Apr 2013 5:03

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 ????

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

Post by LHCTom » Mon 29 Apr 2013 6:30

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 ????
You mean false negatives - but I agree. I seriously doubt the CDC 2T would pick up these species so would be false negatives and you can add Borrelia miyamotoi to the list. The dogma around " There is only one pathogenic species in the US " is quite strong and indicates many otherwise smart people have no imagination or creativity. It reminds me of the 911 commission suggesting "a failure of imagination". The same thing is happening here. Its not just other species ---its widely divergent strains also. And a whole list of other reasons.

I'm a great example of multiple false negatives and they were all different. One lab showed no IgG WB bands, one showed 3 CDC IgG bands and another showed 3 CDC IgG bands but 2 were different. That means if you count total bands found across 2 of the labs, I scored 5/10. But two 3/10's is a negative. I was then tested for C6 at 3 labs and were strong positives. I was ALS culture positive and had the DNA checked by PCR as positive for Bb and sequenced by Eurofin and the partial sequence matched the NCBI Borrelia xxx database. The deniers :evil: on this site need an infusion of imagination. :bonk:
The greater the ignorance, the greater the dogmatism.

Attributed to William Osler, 1902

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

Post by LHCTom » Mon 29 Apr 2013 6:45

Is there a mathematician on this forum who would care to work out the odds or probabilities based on 5 out of 9 possible bands for any given strain versus the 10 that the CDC is implying should be possible for all strains?
The mathematics is not difficult but it means nothing if the data is biased. So far, most studies include only the strains in their geographic area and have not even considered species diversity. The Chinese study above has a reasonable explanation of an approach using some obvious simplifying plus a ROC analysis. But if the data isn't a large sample and doesn't include a strain species diversity that roughly matches the US diversity proportionately and accurately plus the true positives are properly determined, its just garbage in = garbage out. Not one US study has come even close to creating good data. :woohoo:

And its obvious and shows!!!!! For those with a smidge or touch of imagination.
The greater the ignorance, the greater the dogmatism.

Attributed to William Osler, 1902

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