Dr Benjamin Luft GeneChipProteinArray detects Antibodies

Topics with information and discussion about published studies related to Lyme disease and other tick-borne diseases.
Lorima
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Joined: Mon 29 Oct 2007 20:47

Re: Dr Benjamin Luft GeneChipProteinArray detects Antibodie

Post by Lorima » Fri 17 Aug 2012 3:50

Dear Henry,

Regarding your first comment, your resorting to a term like "unreasonably paranoid", substantiates my judgment that I'm not being so. ;-)
No hard feelings, I've gathered that the culture of medicine tolerates this sort of thing. I'd rib you back, but in my field, saying something like that would detract from one's professional reputation. Consequently, I don't have any practice, and would probably get it wrong.

Regarding the politics, very interesting! do tell. Whose group seems to be ahead? Who in the FDA and CDC are being "receptive and supportive" to whom?

Actually, I'd rather they were being discerning and disinterested; a new test that's no improvement on the old one is worse than useless. We'll be stuck with it until its patents expire.

But I agree with Dr. MacDonald that the microarray should be developed; it will be useful when and if the politics gets cleaned up.

The problem with the politics is this: the US medical establishment and its followers are not even making full use of the the antigens specific to Bb that are detected on the current standard western blots.

As you know, the current recommendations actually mandate withholding information about most of the specific bands from the doctors and their patients. From a scientific standpoint, it's a jaw-dropper. We're simply throwing bought-and-paid-for information away, for every patient, every day, in labs all over the country.

So how can detecting more antibodies to more antigens on a chip improve diagnosis, in this climate? The interpretation scheme still can be botched, and predictably will be, unless there's a complete clean-out of the current power players.

I'm happy to say the whole world hasn't been fooled, though: the Chinese CDC has taken the initiative to do their own research, and have come up with recommendations very similar to (dare I say?) ILADS.
http://www.ncbi.nlm.nih.gov/pubmed/21112481
Very simple; report all the bands, report which ones crossreact with other antigens and which ones, conversely, are specific to Bb; judge the results of the blots accordingly. Maybe we can just outsource our Lyme testing to China.

Though I have enough money that I don't mind treating my friends and family to Igenex. I'm thinking of trying Clongen next time, as they send actual pictures of the blots. I think I'll write to all the specialty labs and suggest they do it too.

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

Henry
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Re: Dr Benjamin Luft GeneChipProteinArray detects Antibodie

Post by Henry » Fri 17 Aug 2012 16:13

The CDC used the same type of analysis to derive their criteria for IgM and IgG Western blots.

Lorima
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Re: Dr Benjamin Luft GeneChipProteinArray detects Antibodie

Post by Lorima » Fri 17 Aug 2012 20:35

Could you give me a ref for that (preferably a paper with the actual data analysis, patient selection, etc. details)?
Thanks much,
Lorima
"I have to understand the world, you see."
Richard Feynman

radicale
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Re: Dr Benjamin Luft GeneChipProteinArray detects Antibodie

Post by radicale » Fri 17 Aug 2012 22:41

I think you hit the nail on the head Lorima with your comment regarding the 100% sensitivity claim. This type of sloppy (at best) claim paints academics in a rather negative light. These types of half-truths/lies are rampant in academia. Interestingly enough, it seems that some have said enough is enough
http://blogs.plos.org/everyone/2012/08/ ... nitiative/
PLOS ONE is pleased to announce a collaboration with Science Exchange and figshare in a groundbreaking new project: The Reproducibility Initiative. The initiative aims to help scientists validate their research findings by providing a mechanism for blind, independent replication by experts from Science Exchange’s network of more than 1,000 providers at core facilities and contract research organizations.
After the guidelines were published there was concern among some medical professionals that the criteria should not be used for clinical diagnosis.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC229023/
Recommendation to include OspA and OspB in the new immunoblotting criteria for serodiagnosis of Lyme disease.
E Hilton, J Devoti, and S Sood
Department of Medicine, Long Island Jewish Medical Center, New Hyde Park, New York 11042, USA.

In October 1994, the Second National Conference on the Serologic Diagnosis of Lyme Disease recommended a two-step approach to serological testing. The first step was the performance of an enzyme-linked immunosorbent assay (ELISA); the second step was a confirmatory immunoblot. New criteria for the interpretation of a positive immunoblot were also recommended. The committee decided to omit the 31- and 34-kDa bands (OspA and OspB, respectively) from the choice of bands considered diagnostic for a positive immunoblot. Since we had previously included these in our diagnostic criteria for Lyme disease-positive immunoblots, we reviewed data for all patients attending a Lyme disease center with positive ELISAs and immunoblot assays for Lyme disease from 1 September 1992 to 31 December 1993. The criteria for a positive Western blot (immunoblot) were the presence of 5 of 12 bands, including the 10 recommended by the conference, and the presence of the 31- and 34-kDa protein bands. Of the 136 patients evaluated, 50 were considered to have Lyme disease. Of these 50, 4 (8%) would not have met immunoblot criteria for the diagnosis if the new recommendations were used. Had the 31- and 34-kDa bands been included as part of the diagnostic requirements for immunoblot, these patients would have been included. Although overdiagnosis of Lyme disease appears to be the more frequent problem, our concern is that the exclusion of the 31- and 34-kDa protein bands from the diagnostic criteria may result in the underdiagnosis of Lyme disease by those who would rely too heavily on serological confirmation. The addition of the 31- and 34-kDa bands to those recommended for confirmatory immunoblot should be reconsidered.
In addition, the sensitivity varies depending on who is doing the testing and depending on what they are trying to show. Let's compare two studies with respect to the two-tier testing sensitivity:

Here is a look at a new multiplex assay.
http://www.ncbi.nlm.nih.gov/pmc/article ... =pmcentrez
Multiplex Immunoassay for Lyme Disease Using VlsE1-IgG and pepC10-IgM Antibodies: Improving Test Performance through Bioinformatics
Richard B. Porwancher,1,2,* C. Greg Hagerty,1 Jianqing Fan,3 Lisa Landsberg,2 Barbara J. B. Johnson,4 Mark Kopnitsky,5 Allen C. Steere,6 Karen Kulas,7 and Susan J. Wong7

The Centers for Disease Control and Prevention currently recommends a 2-tier serologic approach to Lyme disease laboratory diagnosis, comprised of an initial serum enzyme immunoassay (EIA) for antibody to Borrelia burgdorferi followed by supplementary IgG and IgM Western blotting of EIA-positive or -equivocal samples. Western blot accuracy is limited by subjective interpretation of weakly positive bands, false-positive IgM immunoblots, and low sensitivity for detection of early disease. We developed an objective alternative second-tier immunoassay using a multiplex microsphere system that measures VlsE1-IgG and pepC10-IgM antibodies simultaneously in the same sample. Our study population comprised 79 patients with early acute Lyme disease, 82 patients with early-convalescent-phase disease, 47 patients with stage II and III disease, 34 patients post-antibiotic treatment, and 794 controls. A bioinformatic technique called partial receiver-operator characteristic (ROC) regression was used to combine individual antibody levels into a single diagnostic score with a single cutoff; this technique enhances test performance when a high specificity is required (e.g., ≥95%). Compared to Western blotting, the multiplex assay was equally specific (95.6%) but 20.7% more sensitive for early-convalescent-phase disease (89.0% versus 68.3%, respectively; 95% confidence interval [95% CI] for difference, 12.1% to 30.9%) and 12.5% more sensitive overall (75.0% versus 62.5%, respectively; 95% CI for difference, 8.1% to 17.1%). As a second-tier test, a multiplex assay for VlsE1-IgG and pepC10-IgM antibodies performed as well as or better than Western blotting for Lyme disease diagnosis. Prospective validation studies appear to be warranted.
Here is another one.
http://www.ncbi.nlm.nih.gov/pubmed/19947857
2-tiered antibody testing for early and late Lyme disease using only an immunoglobulin G blot with the addition of a VlsE band as the second-tier test.
Branda JA, Aguero-Rosenfeld ME, Ferraro MJ, Johnson BJ, Wormser GP, Steere AC.
Source
Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA. branda.john@mgh.harvard.edu
Abstract
BACKGROUND:
Standard 2-tiered immunoglobulin G (IgG) testing has performed well in late Lyme disease (LD), but IgM testing early in the illness has been problematic. IgG VlsE antibody testing, by itself, improves early sensitivity, but may lower specificity. We studied whether elements of the 2 approaches could be combined to produce a second-tier IgG blot that performs well throughout the infection.
METHODS:
Separate serum sets from LD patients and control subjects were tested independently at 2 medical centers using whole-cell enzyme immunoassays and IgM and IgG immunoblots, with recombinant VlsE added to the IgG blots. The results from both centers were combined, and a new second-tier IgG algorithm was developed.
RESULTS:
With standard 2-tiered IgM and IgG testing, 31% of patients with active erythema migrans (stage 1), 63% of those with acute neuroborreliosis or carditis (stage 2), and 100% of those with arthritis or late neurologic involvement (stage 3) had positive results. Using new IgG criteria, in which only the VlsE band was scored as a second-tier test among patients with early LD (stage 1 or 2) and 5 of 11 IgG bands were required in those with stage 3 LD, 34% of patients with stage 1, 96% of those with stage 2, and 100% of those with stage 3 infection had positive responses. Both new and standard testing achieved 100% specificity.
CONCLUSIONS:
Compared with standard IgM and IgG testing, the new IgG algorithm (with VlsE band) eliminates the need for IgM testing; it provides comparable or better sensitivity, and it maintains high specificity.
One really needs to look at the actual data to come to any intelligent conclusions. For the second study the testing was conducted at two locations and the sensitivity depended wildly on where it was. If you had stage II disease and were tested at Massachusetts General Hospital (MGH) the sensitivity of the test would have been 42% while if you were lucky to stumble into Westchester Medical Center (WMC) the sensitivity of the test used would have been 80%! Is this due to a different set of patients and perhaps greater dissemination at WMC:
The proportion of patients with evidence of hematogenous dissemination, as assessed by the number of patients with multiple EM lesions (the only marker that was available in both sets of patients) was higher in the MGH set (19%) than in the WMC set (7%) ( ), which is the opposite of what would be P ! .001 expected if this factor were important in explaining the higher frequency of seroreactivity in the WMC set.
Wait a second, I thought that patients presenting with multiple EM lesions tested off the charts. The authors didn't care to try to explain further, how convenient.

Other interesting discussions regarding selection of appropriate number of specific IgG bands taken from the paper:
With a requirement of 99% specificity, the greatest sensitivity was achieved using a cutoff of 3 of 11 specific IgG bands (18, 23, 28, 30, 39, 41, 45, 58, 66, and 93 kD plus VlsE). However, compared with standard 2-tiered IgM and IgG testing, there was still considerable loss of sensitivity in stage 1 LD.
First of all, what does IgG have to do with stage 1LD!? Furthermore, why would you decrease the sensitivity by requiring two additional bands when you already have a 99% specificity? Is this sound logic? Perhaps for surveillance but definitely not for clinic diagnosis.

Furthermore, if we compare this with the first study we can see that the sensitivity of patients with third stage disease is not 100%.
Attachments
table_western_blot_sensitivity.png
Western Blot Sensitivity
table_western_blot_sensitivity.png (43.07 KiB) Viewed 1140 times
Last edited by radicale on Sat 18 Aug 2012 2:09, edited 2 times in total.

radicale
Posts: 134
Joined: Fri 4 May 2012 16:51

Re: Dr Benjamin Luft GeneChipProteinArray detects Antibodie

Post by radicale » Fri 17 Aug 2012 23:33

What exactly is a gold standard test? How can one be sure it is? here is a relevant look at the testing for another spirochaete.
Lorima wrote:Hello Henry,
Yes, what I'm really wondering is if it would have a chance, politically, of reaching patients even if it were meticulously shown to be superior in every way to the current test.

It's doubtful that it would have such a chance, considering, as you say, that any new test will have to agree with the current test, which will be treated as a gold standard ("100% sensitive in late disease", etc.).
(I believe the current testing algorithm to be fatally flawed, by both scientific and public health standards. I'm a molecular biologist with an impressive CV, who lives in an LD-endemic community. I have enough contact with the medical research and policy community to have observed many of its foibles. Please, let's not propagate an unduly optimistic view of its overall scientific rigor, compared with its other priorities, like reassuring the public, and easing the difficulties of practicing medicine in cases of uncertainty. You sound intellectually sophisticated enough to understand these matters in some detail, if you wish.)

I'm sorry if my parenthetical comment sounds harsh; it's not my intention to gratuitously offend anybody. But I guess people who disagree with us are always somewhat offensive, if only because they're, well, "disagreeable". ;-)

Sincerely, Lorima
http://cid.oxfordjournals.org/content/55/3/322
Fool's Gold: Why Imperfect Reference Tests Are Undermining the Evaluation of Novel Diagnostics: A Reevaluation of 5 Diagnostic Tests for Leptospirosis

Background. We observed that some patients with clinical leptospirosis supported by positive results of rapid tests were negative for leptospirosis on the basis of our diagnostic gold standard, which involves isolation of Leptospira species from blood culture and/or a positive result of a microscopic agglutination test (MAT). We hypothesized that our reference standard was imperfect and used statistical modeling to investigate this hypothesis.

Methods. Data for 1652 patients with suspected leptospirosis recruited during three observational studies and one randomized control trial that described the application of culture, MAT, immunofluorescence assay (IFA), lateral flow (LF) and/or PCR targeting the 16S rRNA gene were reevaluated using Bayesian latent class models and random-effects meta-analysis.

Results. The estimated sensitivities of culture alone, MAT alone, and culture plus MAT (for which the result was considered positive if one or both tests had a positive result) were 10.5% (95% credible interval [CrI], 2.7%–27.5%), 49.8% (95% CrI, 37.6%–60.8%), and 55.5% (95% CrI, 42.9%–67.7%), respectively. These low sensitivities were present across all 4 studies. The estimated specificity of MAT alone (and of culture plus MAT) was 98.8% (95% CrI, 92.8%–100.0%). The estimated sensitivities and specificities of PCR (52.7% [95% CrI, 45.2%–60.6%] and 97.2% [95% CrI, 92.0%–99.8%], respectively), lateral flow test (85.6% [95% CrI, 77.5%–93.2%] and 96.2% [95% CrI, 87.7%–99.8%], respectively), and immunofluorescence assay (45.5% [95% CrI, 33.3%–60.9%] and 96.8% [95% CrI, 92.8%–99.8%], respectively) were considerably different from estimates in which culture plus MAT was considered a perfect gold standard test.

Conclusions. Our findings show that culture plus MAT is an imperfect gold standard against which to compare alterative tests for the diagnosis of leptospirosis. Rapid point-of-care tests for this infection would bring an important improvement in patient care, but their future evaluation will require careful consideration of the reference test(s) used and the inclusion of appropriate statistical models.
Note the number of patient samples used, 1652. That sounds about right.

Lorima
Posts: 914
Joined: Mon 29 Oct 2007 20:47

Re: Dr Benjamin Luft GeneChipProteinArray detects Antibodie

Post by Lorima » Sat 18 Aug 2012 14:34

Good points. Before I respond in detail, I'll wait for Henry to give us the ref(s) for any US CDC work that corresponds to the Chinese CDC study I cited:
Henry wrote:The CDC used the same type of analysis to derive their criteria for IgM and IgG Western blots.
Here's the link and abstract for the Chinese CDC's paper:

http://www.ncbi.nlm.nih.gov/pubmed/21112481

Full text pdf: http://www.besjournal.com/Articles/Arch ... 120607.pdf
(Save a copy for yourself - It's not in PMC, so maybe won't always be available)
Biomed Environ Sci. 2010 Oct;23(5):341-9.

Interpretation criteria for standardized Western blot for the predominant species of Borrelia burgdorferi sensu lato in China.

Jiang Y, Hou XX, Geng Z, Hao Q, Wan KL.
Source
State Key Laboratory for Infectious Diseases Prevention and Control, National Institute of Communicable Disease Control & Prevention, Chinese Center of Disease Control & Prevention, Beijing 102206, China.
Abstract
OBJECTIVE:
Western blotting (WB; immunoblotting) is a widely used tool for the serodiagnosis of Lyme borreliosis (LB), but so far, no generally accepted criteria for its performance and interpretation have been established in China. The present study was designed to determine the criteria for standardized Western blot for the predominant species of Borrelia burgdorferi sensu lato in China, in which WB was produced with strain PD₉₁ as the representative strain attributed to predominant genospecies Borrelia garinii of Borrelia burgdorferi sensu lato.
METHODS:
Approximately 13 bands between 14 and 100 kD were differentiated for strain PD₉₁ by using Gel-Pro analysis software. In a study with 631 serum samples (taken from 127 patients with Lyme borreliosis and 504 controls), all observed bands were documented. To establish criteria for a positive WB result for strain PD₉₁, receiver operating characteristic (ROC) curves were used.
RESULTS:
The following interpretation criteria were recommended:for IgG, at least one band of P83/100, P58, P39, P30, OspC, P17, P66, and OspA; for IgM, at least one band of P83/100, P58, OspA, P30, OspC, P17 or P41. In addition, syphilis, leptospirosis and other related diseases should be excluded when the positive band is P41 in IgM. For IgG criteria, the sensitivity is 73.2%, the specificity is 99.4% and Youden index is 0.726; for IgM criteria, the sensitivity is 50.6%, the specificity is 93.1% and Youden index is 0.437.
CONCLUSION:
Standardization of WB assays is necessary for comparison of results from different laboratories. Moreover, the criteria of other genospecies of Borrelia burgdorferi sensu lato should be determined in the future to complete the criteria of WB for the diagnosis of the Lyme disease in China.
Copyright © 2010 The Editorial Board of Biomedical and Environmental Sciences. Published by Elsevier B.V. All rights reserved.
PMID: 21112481 [PubMed - indexed for MEDLINE]
Their strain, PD91, doesn't express OspB (=P34) (see Fig.1), so it's not on their list of specific bands, but presumably should be for the US, as mentioned in the communication Radicale cited above:
Recommendation to include OspA and OspB in the new immunoblotting criteria for serodiagnosis of Lyme disease.
E Hilton, J Devoti, and S Sood by Hilton, Devoti, and Sood: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC229023/

Henry, I'm looking forward to your reference(s) from the US CDC.

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

Henry
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Re: Dr Benjamin Luft GeneChipProteinArray detects Antibodie

Post by Henry » Sat 18 Aug 2012 19:43

Try this: http://www.cdc.gov/lyme/diagnosistreatm ... t/TwoStep/

There is a telephone number listed at the end of the recommendations memo where you can call for more specific details.

Lorima
Posts: 914
Joined: Mon 29 Oct 2007 20:47

Re: Dr Benjamin Luft GeneChipProteinArray detects Antibodie

Post by Lorima » Tue 21 Aug 2012 3:31

Ah. Yes. [Henry's link leads us back to Dressler et al. 1993, from Steere's lab]

I thought you meant that someone at the CDC had done research on Lyme western blot criteria. The Dressler et al. work was done in Dr. Steere's lab at Tufts.

The citation of Engstrom et al. from U of Minn., whose paper recommends different Western blot criteria, has to do with (1)their recommendation to test again later if the first test is negative and (2) their recommendation to test for both IgG and IgM in early disease. Engstrom also suggests that the Western blot is usually more sensitive than the ELISA, as well as more specific.

My point is, that Henry has brought us back to 1993 for data supporting the current testing protocol recommended by the US's Centers for Disease Control, and it's from one lab - Dr. Steere's.

We might ask how this one study came to determine whether persons infected with Bb would be allowed diagnosis and treatment, in the US, Canada, and Europe, almost 20 years later.

First, we need to understand why the US CDC adopted it. I think the best clue to this is found in Craven et al., 1996:

http://www.ncbi.nlm.nih.gov/pmc/article ... 903216.pdf

This paper shows how the CDC was persuaded to abandon their more sensitive ELISA for a less sensitive but more specific one, further restricted by a very demanding Western blot test.

I'm still working on how to explain in detail what I think this paper tells us about how this consequential, and for many, disastrous event came about. I think I'd better post this first part now, though, before the topic gets stale. Meanwhile, anyone interested might collect and print out the paper.

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

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