1 Paper: Fluorescent Bead-Based Multiplex Assay (For Detection of Bb Antibodies)

I was originally going to write on a paper on Borrelia burgdorferi and lymphadenopathy that has been making the rounds, but the following link was sent to me and I think it deserves more immediate attention:

http://ahdc.vet.cornell.edu/docs/Scopes_2011_02.pdf

Following this link, on page 6 of the document is an interview with Dr. Bettina Wagner, Assistant Professor of Equine Health at Cornell University, on the use of a Lyme disease blood test for dogs which is more accurate than earlier tests.

The test uses a technology known as bead-based multiplex technology, a technology that has been in use for the past decade - but this is the first veterinary diagnostic lab which is using it for Lyme disease.

Key excerpts from the article:

The improved test for Lyme disease in horses and dogs developed by Wagner and her colleagues takes less time, requires smaller samples, and answers more questions about the disease. In the past, diagnosticians had to run several tests to confirm Lyme disease. The multiplex procedure can detect many kinds of antibodies to several different antigens of B. burgdorferi using a single test on a single sample, eliminating the need for separate tests.

[...] Different kinds of antibodies can be found in the body at different stages of infection. The new test can distinguish and measure these differences, giving more information about the timing of the disease. "We can now not only distinguish between infection and vaccination, but also between early and chronic infection stages,” Wagner noted. “That was not possible before. You were able to say whether an animal was infected, but not when it was infected, or how far the infection had developed.”

The test and information it provides can help veterinarians make advanced decisions about treatment. After the long treatment period ends, veterinarians usually conduct follow-up testing to see if it was successful.

I decided I wanted to learn more about Dr. Wagner and if she did any research - as well as to learn more about bead-based multiplex assays.

I also had one question: Why isn't this test available for people, since many have complained that Lyme disease serological tests are not that accurate?

Anyway, I immediately found this abstract (USD $31.50 for full text):

A fluorescent bead-based multiplex assay for the simultaneous detection of antibodies to B. burgdorferi outer surface proteins in canine serum

B. Wagnera, H. Freera, A. Rollinsa, and H.N. Erba
Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA
Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA
Received 28 September 2010; revised 22 November 2010; accepted 2 December 2010. Available online 10 December 2010.

Abstract

Lyme disease is a zoonotic, vector-borne disease affecting humans, dogs, horses and other species. It is caused by infection with spirochetes of the Borrelia burgdorferi sensu lato group which are transmitted to the mammalian host by infected ticks (Ixodes). Exposure to B. burgdorferi is commonly diagnosed by serological testing. The gold standard for the detection of antibodies to B. burgdorferi is a two-step procedure of an ELISA followed by confirmatory Western blotting (WB).

Here, we developed and validated a new bead-based multiplex assay for the detection of antibodies to B. burgdorferi in canine serum which combined the testing by ELISA and WB in a single quantitative test. B. burgdorferi outer surface protein A (OspA), OspC and OspF were expressed in E. coli. The recombinant proteins were coupled to fluorescent beads providing the matrix of the assay.

Two sets of canine sera were used for validation of the multiplex assay. First, sera from 79 dogs with known ELISA and WB results were used to establish the conditions of the assay. These samples were selected to provide similar numbers of pre-tested sera ranging from negative to high positive results and included sera from vaccinated and/or naturally infected dogs. A high correlation was observed for detection of antibodies to B. burgdorferi in the single and multiplex assays (n = 79). Spearman's rank correlations were 0.93, 0.88 and 0.96 for OspA, OspC and OspF, respectively. Second, a total of 188 canine serum samples that were not tested previously were used for further multiplex assay validation. All samples were also blindly analyzed for antibodies to B. burgdorferi antigens by WB. The WB results provided a ‘relative gold standard’ for each antigen and were used to perform a receiver operating curve analysis.

The areas under the curves were 0.93 for OspA, 0.82 for OspC, and 0.89 for OspF. Multiplex assay interpretation ranges for antibodies to all three B. burgdorferi antigens in canine serum were established by likelihood analysis. The diagnostic sensitivities of the individual OspA, OspC and OspF bead-based assays were 83%, 62% and 82%, respectively, and the diagnostic specificities were 90%, 89% and 86%, respectively.

The new multiplex assay provides a sensitive and fully quantitative platform for the simultaneous evaluation of antibodies to B. burgdorferi OspA, OspC and OspF antigens and distinguishes between antibodies that originated from vaccination or natural exposure to B. burgdorferi.'

Keywords: Lyme disease; Dog; Zoonoses; Diagnostic assay; High throughput analysis
Abbreviations: MFI, median fluorescence intensity; Osp, outer surface protein; ROC, receiver operating curve; WB, Western blotting

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The one obvious thing to note about this test is that it can distiguish between antibodies that originated from vaccination or natural exposure to B. burgdorferi.

This is important in the canine model, where dogs are regularly vaccinated for Lyme disease and humans currently are not. However, if a future vaccine for humans is introduced, it will be important to distinguish between these two cases, and be able to treat a patient who had a vaccine who happened to get infected with Borrelia burgdorferi after the vaccination or inbetween booster shots.

One of the controversies over current serological testing for Lyme disease is that a positive result on kDa bands 31 and 34 are not included in the list of CDC surveillance bands to determine a reportable case of Lyme disease - when bands 31 and 34 are OspA and OspB - outer surface proteins which are very specific to Borrelia burgdorferi (Bb).

It has been speculated that the reason these bands were dropped from inclusion in a positive result for Bb is because the original Lymerix vaccine would have created a false positive for these bands in a vaccinated patient taking the test - if so, though, my question is why not just ask the patient if they had the vaccine, firstly - and secondly, why not treat the patient based on a clinical diagnosis anyway?

For those of you reading along who are not familiar with this issue of omission of highly sensitive bands from the Lyme disease blood test, I recommend checking out this paper, Recommendation To Include OspA and OspB in the New Immunoblotting Criteria for Serodiagnosis of Lyme Disease (.pdf), by Hilton, Devoti, and Sood. Here's a notable excerpt from that:

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.

What happened with this? They are still not included in the current test for defining a positive result. So who knows how often people are underdiagnosed because of this.

Anyway... putting aside the patient community's reports of a class action lawsuit against GSK (who put Lymerix on the market in the late 1990s) and its subsequently getting pulled off the shelves, the Lymerix vaccine was not 100% effective against all strains of Bb and was a costly battery of vaccinations for one not covered by insurance (USD $250).  And those vaccinated could still get Lyme disease, much like those vaccinated for the flu could still get the flu. It was just supposed to provide protection that improved your odds against infection.

Now getting back to the question I asked earlier about human serological tests based on this same bead multiplex technology used for canine Lyme disease tests?

Well, they do exist. With a little time and the magic of google, I found that Luminex recombinant antigen fluorescent bead multiplex assays for human detection of Bb are being sold in Germany, with 96 tests to a kit, at Mikrogen Diagnostik.

There is a detailed page on this here: http://www.mikrogen.de/deutsch/deutschland/nc/produkte/testsysteme/testsystem/borrelia-igg.html

Two references are provided in relation to this test, which I have yet to review:

1) Wilske B, Zöller L, Brade V, Eiffert H, Göbel UB, Stanek G, Pfister HW and: MIQ 12 Lyme disease. In quality standards in the microbiologically-infectious diseases diagnostics. H. Mauch, and R. Lütticken, eds. Munich, Germany, Urban & Fischer Verlag, 2000, pp. 1-59

2) DIN 58969-44: Medical microbiology - Serological and molecular diagnosis of infectious diseases - Part 44: Immunoblot (IB) Special requirements for the detection of antibodies to Borrelia burgdorferi.

Who is Luminex? They're an American company, and you might have heard about them in the news recently because their xTag GPP test was used for triage of German patients with E. coli infections. Yes, those seriously bad E. coli infections, that had everyone in Germany wondering if they were going to be eating endless plates of bratwurst and drinking endless liters of beer with no vegetables to be seen.

If you aren't familiar with Luminex themselves, then you may be more familiar with their partners, who range from the well-known Labcorp of America through the less well-known ZEUS Scientific, who put out that AtheNA Multi-Lyte® Test System Lyme disease test I mentioned in the past.

But getting back to the human fluorescent tests... How good are they?  I include the below charts from Mikrogen Diagnostik for you to decide:

It should be noted that the above data are for a European test, thus the measurements will differ from an American Bb test. I tried to find Lyme disease tests directly from Luminex on their web site where they list all infectious disease test kits they provide, but Lyme disease is not one of them. They do, however, note that their spin-off company, Rules-Based Medicine (RBM), works on Lyme disease testing - yet there is no specific Lyme disease assay listed on their product page - although there are a lot of biomarker tests available. By the way, the most recent word on RBM is that as of June 1, it was acquired by a company known as Myriad Genetics Inc. - so who knows what will happen with its product line now.

I'm still looking for Luminex clinical diagnostic tests for Lyme disease that are being used in the US - but even if I don't find any, it's useful to know researchers are using Luminex assays here for Bb studies, such as in this recent study by Steven Norris:

http://aem.asm.org/cgi/content/abstract/77/4/1483

Understand that you will find a number of studies which rely on multiplex assays already out there - but most of them are not aimed towards clinical diagnosis.

As an addendum to this, I managed to find another company's multiplex IgM Borrelia assay which relies on Luminex technology - so I have to wonder who holds the patent on this technology and who is paying royalties on it. The company is Multimetrix, and you just may want to look at their pdf on Multimetrix multiplex IgM Borrelia assays: http://www.multimetrix.com/fileadmin/pdf/borrelia-test_en.pdf

On this handout, it is explained how the Multimetrix multiplex test works (click to view zoomed-in image):

In addition to this, if you are a microbiology geek, here is a video of a live demonstration from end-to-end of the Multiplex assay mentioned [Time: 9:49]:



I warn you, though, that it might bore you to tears unless you are evaluating this test for use in your own lab (even then, it still might - it's not like Bio-Rad product ads, that's for sure).

So, this is the news on this sort of assay. Where is the US version for humans? Where is this test currently being used in Europe? These are questions I have yet to answer after this preliminary research.

Other than this, I keep wondering why it is clinical diagnosis is relying on ELISA and not using ELISPOT instead... Any takers?

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