IGeneX changes interpretation criteria for positive serology

[dlf]

I am glad to see that IGeneX had the integrity to determine why the specificity problems appeared in the Fallon study, innovate to try to solve them and then publish this:

http://austinpublishinggroup.com/chroni ... id1009.php#

Shah JS, Du Cruz I, Narciso W, Lo W and Harris NS. Improved Sensitivity of Lyme disease Western
Blots Prepared with a Mixture of Borrelia Burgdorferi Strains 297 and B31. Chronic Dis Int. 2014;1(2): 7.

Received:October 28, 2014;Accepted:December 08, 2014;Published: December 10, 2014

Abstract

Testing for Borrelia burgdorferi (BB), the spirochetal agent of Lyme disease, is problematic due to poor sensitivity of commercially available serological tests. We evaluated an in-house Western Blot (WB) prepared with a mixture of two strains of BB, B31 and 297. Sensitivity and specificity of IgG and IgM testing using the two-strain WB was compared to testing with a commercial single-strain WB, and interpretation of the in-house WB using in-house criteria was compared to interpretation using criteria recommended by the Centers for Disease Control and Prevention (CDC). A total of 364 control and patient sera (including 88 from treated patients with confirmed Lyme disease) were tested. The sensitivity of the combined IgG and IgM commercial WB using CDC criteria was 77.1%. When the in-house IgG and IgM WB and CDC criteria were used, the combined sensitivity improved to 88.6 %, while use of the in-house IgG and IgM WB and in-house interpretation criteria resulted in a combined sensitivity that increased to 97.1%. Using CDC criteria, the specificity of the in-house IgG and IgM WB was 100% and 97.1%, respectively; using in-house criteria; the specificity was 95.3% and 93.1% respectively. By removal of patients who reacted to band 31kDa but tested negative for antibodies to recombinant OspA antigen, in-house IgG and IgM WB specificity increased to >97%. Based on these results, we conclude that the use of the two-strain IgG and IgM WB and in-house interpretation criteria significantly increased the combined sensitivity of the test without significant loss of specificity. In patients with indeterminate WB results, a confirmation test with recombinant OspA is recommended.

<snip>

Antibodies to the OspA antigen banding at 31kDa is present in vaccinated patients and in late stage Lyme disease [28,29]. A large number of serum samples from patients with symptoms of Lyme disease have only bands at 31kDa and 41kDa on either IgG or IgM WB. We reviewed results of Osp A confirmation test results performed on 500 patients with bands 31 and 41kDa on IgG and/or IgM WBs. About 50% of the patients were positive by the Osp A confirmation test. The negative patient samples probably had cross-reacting antibodies to a non-specific BB protein that co-migrates with Osp A at 31kDa. While the truly positive samples would be missed if band 31kDa is not included, the confirmation test was able to resolve a large number of false-positive results. We also observed that 30% of the patients with IgM antibodies to EBV had band 93kDa present with or without band 31kDa on the IgM WB. Removal of band 93kDa from IgM WB interpretive criteria did not affect sensitivity of IgM WB. Thus, the interpretive criteria for Lyme WB have been changed. The in-house WB is considered positive if two bands from the following six bands are present: 23, 31,34, 39, 41 and 93kDa, with the following exception: indeterminate if only bands 31 and 41kDa or 31 and 93kDa are present; and IgM WB is considered negative if only bands 41 and 93kDa are present.

[ChronicLyme19]

Very glad to see they followed up on this as well.

Phew, does not invalidate my results

Interesting to note a lot of late stagers only show bands at 31 and 41.

[ChronicLyme19]

So I wonder what other disease is causing the extra 31dKa bands to show up. That would be neat to know.

I find this part really intriguing:

In patients with late Lyme disease, antibodies to OspA are present [27]. In a prospective study performed in-house on over 400 PCR positive patients with late-stage Lyme disease, 71% had a positive WB (interestingly, 43% of these patients only had a positive IgM WB).

Why would such a large portion of late stage patients not be positive on IgG and only on IgM? (I can only give my own anecdote that as I got sicker with Lyme, my total IgG levels tanked. That doesn't necessarily imply the Lyme caused those to tank tho. I could have gotten sicker because I had lower levels.)

This was interesting to see too. Looks like it's good for Europe as well as the US.

"All 17 sera received from the European patients were positive by the in-house WB."

[Lorima]

Good for Igenex. That's one clue by which you can tell real science from pseudoscience; real science progresses.
My patients each have a bunch of IgM bands; no chance they'll ever look negative. But very skimpy IgG.

CL19 wrote:

Why would such a large portion of late stage patients not be positive on IgG and only on IgM?

That's what Nicole Baumgarth is working on. I called her up a couple of years ago, and she said that was her interest. We at LNE discussed her latest paper on it, last fall:

http://www.lymeneteurope.org/forum/view ... 3&start=10

This is pretty arcane stuff; I just remember that Bb is, indeed, messing up normal class switching from IgM to IgG. And that this may be part of its mechanism for avoiding being wiped out by the immune system of the host. Bb has to stick around in the mouse until the next tick bite, and that's hard to do; mice are great at fighting off infections, in general. Hiding in poorly perfused tissue probably isn't enough to ensure Bb survival.

I need to re-read the paper, but I think it's anti-Bb specific; it's not that they don't make IgGs to other microbes. Bb needs the mouse to stay alive until the next tick bite. However, an immunologist friend of mine once said "You can get along without your B cells; but without your T cells, you're screwed." She focuses on T cells, and was exaggerating; but I think there is a bit of truth there, that I found surprising. Antibodies are easier to understand, than T cell defenses; but that doesn't mean they are more important.

My family LD patients don't seem to have very impaired immunity to other microbes, if any. I don't think one needs to have general immune system problems, to get sick long-term from Bb infection. Bb evolved to survive. Humans are an accidental host, and our longevity may be what's causing the problem. Bb isn't supposed to make the host sick (unlike, say, the cholera bug), and it seems it doesn't do much to short-lived rodents and deer. It may just be that hosting Bb for years or decades, in our tendons, ligaments, and brains, isn't that good for us. It's not that good for Bb either; no chance to move into a new mouse or bird and get passed around into new territory. Not that the individual Bb cares. It's like a houseguest that won't leave, stays way too long, and eventually gets disruptive.

[Claudia]

I find this part really intriguing:

In patients with late Lyme disease, antibodies to OspA are present [27]. In a prospective study performed in-house on over 400 PCR positive patients with late-stage Lyme disease, 71% had a positive WB (interestingly, 43% of these patients only had a positive IgM WB).

Why would such a large portion of late stage patients not be positive on IgG and only on IgM? (I can only give my own anecdote that as I got sicker with Lyme, my total IgG levels tanked. That doesn't necessarily imply the Lyme caused those to tank tho. I could have gotten sicker because I had lower levels.)

Because of my Lyme patient's WB history I am also very interested in the topic of IgM and it's meaning, or lack of meaning.

Several years ago I started a thread related to this: "Maintaining IgM Antibodies for Years, Poor Prognosis in LD" http://www.lymeneteurope.org/forum/view ... f=6&t=3345.

It contains some good discussion and studies with historical content, but also, unfortunately, some ridiculous discussion interference. If you can slog through the pages and separate the wheat from the chaff you might find some things of interest on it too.

I think I'll also bump the old thread hoping for some new input and information.

[ChronicLyme19]

That's what Nicole Baumgarth is working on. I called her up a couple of years ago, and she said that was her interest.

Yeh, I've read through most of her papers I can get a free copy of. It's about the only research I can find so far that would help explain how Lyme screws up your immune system. Other than that I only have anecdotal evidence from my own case, or people on here with immune deficiencies kicking in after tick bites, or stories from my LLMDs that people with Lyme tests who's IgGs light up like xmass tree beat Lyme much more easily than those of us who only show positive IgMs who seem to be stuck with it. I'd be nice to know what types of antibody class switching that gets screwed up. Is it just lyme, is it whole classes, or does it shut down larger chunks of the immune system? There seems to be strong evidence toward at least borrelia antibodies being prevented from forming, but I think I remember the last paper they put out also said it prevented a flu vaccine from working too. (I'll try to dig that one up again).

[Martian]

In this study a total of 364 control and patient sera were tested:

• 88 from treated patients with confirmed Lyme disease
• 276 sera from patients with no history of Lyme disease

29 sera from patients with no history of Lyme disease gave a positive result with use of the IGeneX in-house IgG and IgM WB and in-house interpretation criteria. That's a combined specificity of (276-29)/276 = 89,5%.

Quote from the full text:

29 sera from patients with no history of Lyme disease (16 positive on IgM WB, 10 on IgG and three on IgM and IgG) gave a positive result with the in-house WB criteria, whereas only eight of these 29 subjects gave a positive IgM result by CDC criteria. This included one patient with antibodies to Ehrlichia, three with antibodies related to autoimmune diseases, seven with antibodies to syphilis by Rapid Plasma Reagin (RPR) test, 15 with antibodies to viruses and three controls. Thus the specificity of the in-house IgG and IgM WB were 95.3% and 93.1% respectively. By CDC criteria, the specificity of IgG and IgM WB was 100 % and 97.1% respectively.

OspA WB confirmation

Of the 29 patients with no Lyme-like symptoms, 20 had a band at 31kDa on WB; 19 however, did not react with the recombinant OspA antigen strip. For these 19 patients, the band present at the same position as OspA was not BB specific. This suggests that a non-specific antibody present in the sera is binding to a protein from the cell lysate that co-migrates at the same position as OspA. One serum (IgG and IgM positive) with antibodies to EBV that had bands at 23, 31, 41 and 58kDa, also had antibodies to OspA antigen. Thus, this sample was considered positive for BB-specific antibodies. After removal of the OspA-negative 31kDa-positive samples and “one true positive” there were still 13 samples (eight IgM and five IgG) that were considered to be false positive samples. This included serum from one patient with autoimmune disease, four sera from patients with antibodies to syphilis by RPR test, seven sera from patients with antibodies to viruses and one normal control serum. Thus the in-house WB specificity improved from 95.3% to 98.2% for IgG WB and from 93.1% to 97.1% for IgM WB if the OspA confirmation test was included for any sample that was positive with 31kDa band and one other band used in the in-house criteria of interpretation.

So, with addition of the confirmation test for OspA for any sample that had a band at 31kDa there were still 13 samples that were considered to be false positive. That's a combined specificity of (276-13)/276 = 95,3%.

But what is the effect of the OspA confirmation test on the sensitivity with use of the in-house IgG and IgM WB and in-house interpretation criteria? That is not made clear, but in the discussion section it says:

Antibodies to the OspA antigen banding at 31kDa is present in vaccinated patients and in late stage Lyme disease [28,29]. A large number of serum samples from patients with symptoms of Lyme disease have only bands at 31kDa and 41kDa on either IgG or IgM WB. We reviewed results of Osp A confirmation test results performed on 500 patients with bands 31 and 41kDa on IgG and/ or IgM WBs. About 50% of the patients were positive by the Osp A confirmation test. The negative patient samples probably had crossreacting antibodies to a non-specific BB protein that co-migrates with Osp A at 31kDa. While the truly positive samples would be missed if band 31kDa is not included, the confirmation test was able to resolve a large number of false-positive results.
(...)

In patients with late Lyme disease, antibodies to OspA are present [27]. In a prospective study performed in-house on over 400 PCR positive patients with late-stage Lyme disease, 71% had a positive WB (interestingly, 43% of these patients only had a positive IgM WB). Of these patients, 67% had bands at 31kDa (confirmed as OspA) and 41kDa only on the WB (unpublished data). This data clearly demonstrates the importance of antibodies to OspA in latestage Lyme disease. Thus, any serum sample with an indeterminate WB result (except from vaccinated patients) should be tested with recombinant Osp A WB.

So, with addition of the OspA confirmation test for any sample that had a band at 31kDa the sensitivity with use of the in-house IgG and IgM WB and in-house interpretation criteria is expected to drop considerably.

Also see study "A comparison of Lyme disease serologic test results from four laboratories in patients with persistent symptoms after antibiotic treatment" by Fallon, Brenner, et al. (2014) that includes the IGeneX laboratory.

[ChronicLyme19]

Does anyone know if any of the other borrelia species have band that show up at 31 kDa, like does borrelia miyamotoi for example?

[duncan]

Why were those controls without a history of Lyme that tested positive assumed to be false positives?

[Martian]

Note that the IGeneX website doesn't mention the changed interpretive criteria for the western blot. Further, I found out from discussions on flash.lymenet.org that the 31 Kda confirmation test of IGeneX isn't something new, but already exists since at least 2008. This indicates that the specificity problems of band 31 kDa in the western blot were already known for years. The following information is currently found on the IGeneX website.

Source: http://www.igenex.com

Home > Lyme Disease > Western Blot

About the IgG WESTERN BLOT:

The CDC/ASTPHLD criteria for positive results are 5 of the following 10 antigenic bands: 18 kDa, 23-25 kDa (Osp C); 28 kDa, 30 kDa, 39 kDa; 41 kDa, 45 kDa, 58 Kda, 66 KDa, and/or 83-93 kDa. IGeneX criteria for positive result is 2 of the following 6 bands: 23-25kDa, 31 kDa (Osp A), the 34 kDa (Osp B), 39 kDa, 41kDa and/or the 83-93 kDa. 31kDa and 34kDa antigens are included to the criteria due to their importance in the recurrent and/or persistent disease period. IGeneX criteria of is 96% specific for exposure to B. burgdorferi.

About the IgM WESTERN BLOT:

The antibody specificities of importance for the IgM blot are the same as those for the IgG blot. The CDC/ASTPHLD criteria for positive results are 2 of the following 3 antigenic bands: 23-25 kDa (Osp C); 39 kDa; and/or 41 kDa. IGeneX adds the 31 kDa (Osp A), the 34 kDa (Osp B) (with the argument that these two antigens were used for the vaccines and therefore, antibodies to these antigens are of importance) and/or the 83-93 kDa to the criteria due to their importance in the recurrent and/or persistent disease period. IGeneX criteria of 2 bands is 96% specific for exposure to B. burgdorferi.

Home > Lyme Disease > 30-31 Kda Confirmation

30-31 KDA CONFIRMATION

31 kDa Epitope Test

The Lyme IgG or IgM 31kDa Epitope test is a qualitative immunoblot assay that determines whether the 31kDa band present on a Lyme IgG or IgM Western blot is due to B. burgdorferi specific antibody or not. B. burgdorferi specific epitope 31kDa antigen is denatured and separated by SDS polyacrylamide gel electrophoresis, blotted onto nitrocellulose membranes and cut into strips for the use of Lyme 31 kDa Epitope Test. It is known that Western blots, especially IgM, can give false positive results with some viral and bacterial infections. This test would be very useful to rule out those false positives.


Test # : 488
Description: 31 kDa Epitope Test - IgM*
Specimen: Positive 31 kDa sample previously tested by Western blot at IGeneX


Test #: 489
Description: 31 kDa Epitope Test - IgG**
Specimen: Positive 31 kDa sample previously tested by Western blot at IGeneX


*Not available for NY Residents

For many years we have heard in Lymeland saying that "bands 31 kDa and 34 kDa are so specific for the Lyme spirochete that they were included in a Lyme vaccine!".

But apparently band 31 kDa in the western blot of IGeneX, the best lab according to Lymeland USA, isn't so specific after all. It's actually doing quite poor. So, another Lymeland myth busted.