Lyme neuroborreliosis test improvements

Topics with information and discussion about published studies related to Lyme disease and other tick-borne diseases.
RitaA
Posts: 2768
Joined: Thu 1 Jul 2010 8:33

Lyme neuroborreliosis test improvements

Post by RitaA » Sat 25 Aug 2012 1:18

I'll have to do a bit more digging, but does anyone here happen to know if the recommended improvements/changes below have been put into effect yet? It's been almost 9 years since the first suggestion was published, and four years since the last one:

http://www.ncbi.nlm.nih.gov/pubmed/14648148
J Neurol. 2003 Nov;250(11):1318-27.

Diagnosis of Lyme neuroborreliosis with antibodies to recombinant proteins DbpA, BBK32, and OspC, and VlsE IR6 peptide.

Panelius J, Lahdenne P, Saxén H, Carlsson SA, Heikkilä T, Peltomaa M, Lauhio A, Seppälä I.

Source

Haartman Institute, Dept. of Bacteriology and Immunology, University of Helsinki, 21, 00014, Helsinki, Finland.

Abstract

Three recombinant antigens, decorin binding protein A (DbpA), BBK32, and outer surface protein C (OspC), and IR(6) peptide of borrelial VlsE protein, were evaluated for the diagnosis of neuroborreliosis (NB), using cerebrospinal fluid (CSF) and serum samples from 89 patients. Their performances in enzyme-linked immunosorbent assay (ELISA) were compared with that of commercial flagella antigen. IgG ELISAs were performed with three variants of each recombinant antigen originating from Borrelia burgdorferi sensu stricto, B. afzelii and B. garinii, and with the IR(6) peptide. IgM antibodies were analysed against OspC and flagella. Of the patients whose CSF contained elevated anti-flagella IgG antibodies, 93% were positive for at least three of the new antigens. Of those with negative or borderline CSF anti-flagella antibodies, 51% were positive for three new antigens. Antibodies to BBK32 were detectable mainly in early disease. Antibodies to DbpA and IR(6) were observed in early and late NB. The use of the new antigens at presentation of the disease improved the laboratory diagnosis of NB. In IgG ELISAs, the diagnostic sensitivity of assays with the new antigens was between 75 and 88%, but was only 52% with the flagella antigen. The discriminatory power between patient and control samples appeared better in the CSF than in the serum. We suggest that assessment of CSF antibodies to at least two antigens, using either flagella and one of the new antigens or two of the new antigens, would improve the current diagnostic yield of NB.

PMID:
14648148
[PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov/pubmed/16094226
Pediatr Infect Dis J. 2005 Aug;24(8):709-12.

New antigens for serologic diagnosis of neuroborreliosis in children.

Heikkilä T, Saxen H, Seppälä I, Lönnqvist T, Sillanpää H, Lahdenne P.

Source

Hospital for Children and Adolescents, University of Helsinki, Finland.

Abstract

OBJECTIVE:

To evaluate serology with novel Borrelia-specific protein or peptide antigens in the laboratory diagnosis of neuroborreliosis (NB) in children.

METHODS:

The performance of enzyme-linked immunosorbent assays with several recombinant borrelial protein antigens and invariable region 6 synthetic peptide antigen and of a commercial enzyme-linked immunosorbent assay with the flagella antigen were evaluated in the serodiagnosis and follow-up of children with clinical suspicion of NB. Serum samples were obtained from 20 children with neurologic symptoms indicative of NB. The patients were retrospectively divided into 2 groups based on the laboratory tests at presentation indicating definite (n = 7) or probable (n = 13) NB.

RESULTS:

In addition to cerebrospinal fluid (CSF) lymphocytic pleocytosis and CSF antiflagella antibodies, all 7 patients with definite NB had serum IgG antibodies to at least 2 of the 3 novel antigens at presentation. The 13 patients with probable NB had variable laboratory findings: CSF pleocytosis (n = 7), CSF antiflagella IgM antibodies (n = 4), serum antiflagella IgM and/or IgG antibodies (n = 10). Of these 13 patients, 7 had serum IgG antibodies to 2 of the 3 novel antigens at presentation. During long term follow-up, serum anti-invariable region 6 antibodies disappeared.

CONCLUSIONS:

The present study suggests that assessment of serum antibodies to a panel of Borrelia-specific antigens could improve the laboratory diagnosis of NB at presentation.

PMID:
16094226
[PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov/pubmed/18536620
Pediatr Infect Dis J. 2008 Jul;27(7):605-12.

Improved laboratory diagnostics of Lyme neuroborreliosis in children by detection of antibodies to new antigens in cerebrospinal fluid.

Skogman BH, Croner S, Forsberg P, Ernerudh J, Lahdenne P, Sillanpää H, Seppälä I.

Source

Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköoping University, Sweden.

Abstract

BACKGROUND:

Laboratory diagnostics in Lyme neuroborreliosis need improvement. We hereby investigate 4 new recombinant or peptide Borrelia antigens in cerebrospinal fluid in children with neuroborreliosis to evaluate their performance as diagnostic antigens.

METHODS:

An enzyme-linked immunosorbent assay was used to detect IgG antibodies to recombinant decorin binding protein A (DbpA), BBK32, outer surface protein C (OspC), and the invariable region 6 peptide (IR6). The recombinant antigens originated from 3 pathogenic subspecies; Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi sensu stricto. Cerebrospinal fluid and serum from children with clinical features indicative for neuroborreliosis (n = 57) were analyzed. Classification of patients was based on clinical symptoms and laboratory findings. Controls were children with other neurologic diseases (n = 20) and adult patients with no proven infection (n = 16).

RESULTS:

Sensitivity for DbpA was 82%, for BBK32 70%, for OspC 58% and for IR6 70%. Specificities were 94%, 100%, 97%, and 97%, respectively. No single antigen was superior. When new antigens were combined in a panel, sensitivity was 80% and specificity 100%. The reference flagella antigen showed a sensitivity of 60% and a specificity of 100%. Over all, the B. garinii related antigens dominated.

CONCLUSIONS:

Recombinant DbpA and BBK32 as well as the peptide antigen IR6 perform well in laboratory diagnostics of neuroborreliosis in children. New antigens seem to improve diagnostic performance when compared with the routine flagella antigen. If different antigens are combined in a panel to cover the antigenic diversity, sensitivity improves further and a specificity of 100% can be achieve.

PMID:
18536620
[PubMed - indexed for MEDLINE]

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

Re: Lyme neuroborreliosis test improvements

Post by radicale » Sat 25 Aug 2012 1:25

I wonder why these types of improvements are still not being taken advantage of in clinical practice. All make claims of significantly improved sensitivity, and even specificity. It is odd, the science appears to be there but something is getting in the way of their use.

RitaA
Posts: 2768
Joined: Thu 1 Jul 2010 8:33

Re: Lyme neuroborreliosis test improvements

Post by RitaA » Sun 26 Aug 2012 7:52

radicale wrote:I wonder why these types of improvements are still not being taken advantage of in clinical practice. All make claims of significantly improved sensitivity, and even specificity. It is odd, the science appears to be there but something is getting in the way of their use.
I was hoping to find out whether or not some of the suggestions/recommendations have already been incorporated into current testing methods. I haven't found any answers yet, but I did come across another interesting article abstract during my search:

http://www.ncbi.nlm.nih.gov/pubmed/15679490
Clin Microbiol Infect. 2005 Feb;11(2):147-50.

Analysis of Borrelia burgdorferi IgG antibodies with a combination of IgG ELISA and VlsE C6 peptide ELISA.

Jansson C, Carlsson SA, Granlund H, Wahlberg P, Nyman D.

Source

Laboratory Department, Aland Central Hospital, Aland, Finland.

Abstract

Enzyme-linked immunosorbent assays (ELISAs) were used to detect antibodies to the C6 peptide of the Borrelia burgdorferi VlsE protein and a selection of B. burgdorferi IgG antigens, separately and as a combination, in 355 serum specimens from blood donors and patients. Western immunoblotting was used as the reference method. The sensitivity of the combined analysis of IgG antigen and C6 peptide analysis was markedly superior to those of the separate analyses. When the C6 peptide and IgG results were concordant, the customary confirmatory Western immunoblotting assay could be omitted, thus reducing the time and cost of analysis.

PMID: 15679490 [PubMed - indexed for MEDLINE]
If I'm understanding the abstract correctly, the combined analysis would provide a more accurate screening test that might even eliminate the need for a much more expensive and labour-intensive confirmatory WB -- at least in some cases. I'd like to know if even this has become standard practice anywhere in the world.

Edited to add:

To add to my confusion, here's a published article by Steere et al:

http://cid.oxfordjournals.org/content/47/2/188.long
Clin Infect Dis. (2008) 47 (2): 188-195. doi: 10.1086/589242

Prospective Study of Serologic Tests for Lyme Disease

Allen C. Steere, Gail McHugh, Nitin Damle, and Vijay K. Sikand

Author Affiliations

Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy and Immunology, Massachusetts General Hospital, Harvard Medical School, Boston

Abstract

Background. Tests to determine serum antibody levels—the 2-tier sonicate immunoglobulin M (IgM) and immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and Western blot method or the IgG of the variable major protein-like sequence-expressed (VlsE) sixth invariant region (C6) peptide ELISA method— are the major tests available for support of the diagnosis of Lyme disease. However, these tests have not been assessed prospectively.

Methods. We used these tests prospectively to determine serologic responses in 134 patients with various manifestations of Lyme disease, 89 patients with other illnesses (with or without a history of Lyme disease), and 136 healthy subjects from areas of endemicity and areas in which the infection was not endemic.

Results. With 2-tier tests and the C6 peptide ELISA, only approximately one-third of 76 patients with erythema migrans had results that were positive for IgM or IgG seroreactivity with Borrelia burgdorferi in acute-phase samples. During convalescence, 3–4 weeks later, almost two-thirds of patients had seroreactivity with the spirochete B. burgdorferi. The frequencies of seroreactivity were significantly greater among patients with spirochetal dissemination than they were among those who lacked evidence of disseminated disease. Of the 44 patients with Lyme disease who had neurologic, heart, or joint involvement, all had positive C6 peptide ELISA results, 42 had IgG responses with 2-tier tests, and 2 patients with facial palsy had only IgM responses. However, among the control groups, the IgG Western blot was slightly more specific than the C6 peptide ELISA. The differences between the 2 test systems (2-tier testing and C6 peptide ELISA) with respect to sensitivity and specificity were not statistically significant.

Conclusions. Except in patients with erythema migrans, both test systems were sensitive for support of the diagnosis of Lyme disease. However, with current methods, 2-tier testing was associated with slightly better specificity.

Henry
Posts: 1108
Joined: Thu 10 Nov 2011 18:49

Re: Lyme neuroborreliosis test improvements

Post by Henry » Mon 27 Aug 2012 15:55

RitaA: The FDA recently approved this Lyme Disease Line Immunoassay developed by Gold Standard Diagnostics: http://www.gsdx.us/index.php/featured-p ... -lyme-line

If you click on the PDF results for the IgM and IgG blots, you will see the criteria and data upon which the criteria are based. Note that bands 31 and 34 kDa are not recognized as having diagnostic significance. When you look at the data got the IgM and IgG blots, compare at the percentages that are associated with early, disseminated, and late Lyme disease. I believe they are derived from comparisons involving >400 specimens.

RitaA
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Joined: Thu 1 Jul 2010 8:33

Re: Lyme neuroborreliosis test improvements

Post by RitaA » Mon 27 Aug 2012 19:30

Thanks, Henry. There's a wealth of information contained in the links you provided.

I was reminded that a number of infections/diagnoses may produce false positive results, although that didn't appear to happen all that frequently with the specimens tested by this test manufacturer:
Cross Reactivity

A cross reactivity study was performed on 215 specimens known to contain potentially cross reactive antibodies to B.burgdorferi. Sera from patients with infections and sera from patients with diagnoses that can be confused with the late manifestations of Lyme disease were tested. The results are summarized in the following table:

[The table includes:

Tick-borne Relapsing Fever / Rickettsial Diseases
Treponemal Infections
Ehrlichiosis
Babesiosis
Leptospirosis
Parvovirus B19
Epstein-Barr Virus
Cytomegalovirus
H. pylori
Fibromyalgia
Rheumatoid Arthritis
Herpes Simplex Virus
Varicella Zoster Virus
Autoimmune Disease]

Two of the 20 Babesiosis samples and one of the 23 Tick-borne relapsing fever / Rickettsial Disease specimens were positive on the Gold Standard Diagnostics B. burgdorferi B31 IgG Line Blot Test. These samples when tested on the predicate device also gave positive results.
No single test should be considered 100% accurate, even according to Gold Standard Diagnostics:
Limitations

1. This assay should be used to test human serum specimens which have been found to be positive or equivocal using an EIA or IFA test procedure. It should not be performed as a primary screening test.
2. A negative result does not completely exclude the possibility of an infection. The sample may have been taken before the appearance of IgG/IgM antibodies, or the antibody titer exists below the detection limit of the test.
3. The treatment of the patients with antibiotics during the early stage of the disease may lead to a suppression of the immune response, so that no specific antibodies may be detected.
4. The diagnosis of Lyme disease must include careful clinical evaluation and should not be based only on detection of antibodies to B. burgdorferi.
5. A positive IgG/IgM test result indicates past exposure and does not necessarily indicate a current infection with B. burgdorferi.
6. The continued presence or absence of antibodies to B. burgdorferi cannot be used to determine the success or failure of therapy.
7. In rare cases a patient serum may show dark background with white bands (inverse bands) which is considered an invalid result and the Line Blot cannot be used in such cases.
Interestingly enough, 25% of people without Lyme disease test positive for IgG band 41 in non-endemic areas, with the percentage increasing to 33% in endemic areas. Unless I'm mistaken, that means approximately one quarter to one third of the population (locations not specified) will test positive for flagella -- which could be from the spirochete associated with dental plaque or other spirochetal infections.

Here's what Gold Standard Diagnostics has to say about Band 31:
You may see the OspA band at 31kDa. Note that it does not have any diagnostic relevance for this test. A single OspA band is an indicator of prior vaccination.
Maybe this is a silly question, but should a positive band 31 result always be ignored -- even in a person who is exhibiting signs/symptoms of Lyme disease and who has never been vaccinated? Is there some other logical explanation for a positive band 31 in a person who hasn't been vaccinated? And yes, I do ask a lot of questions because I'm curious by nature. :)

Henry
Posts: 1108
Joined: Thu 10 Nov 2011 18:49

Re: Lyme neuroborreliosis test improvements

Post by Henry » Tue 28 Aug 2012 0:10

RitaA: It has been established that the 31kDa protein (OspA) is expressed by Borrelia almost exclusively in the midgut of ticks and plays a major role in its adherence to that site. When an infected tick takes a blood meal, either the temperature of the blood -- or some component present in blood-- causes OspC (band 23 kDa) to be expressed in place of OspA. This is enables Borrelia to migrate from the midgut to the salivary glands where it then can be transmitted to the mammalian host. This whole process takes about 36-48 hours, which is why an infected tick must be attached for that length of time before it can transmit infection.

Although OspC is produced abundantly after infection, small amounts of OspA may still be present small amounts, but not enough to generate a detectable antibody response; however, which under certain conditions (e.g. during an inflammatory response to Lyme arthritis when there are lots of inflammatory cytokines produced) some antibody to OspA is produced and can be detected, but not in all patients. Although, the amounts of OspA antibody made are still rather low but detectable, antibodies to the other more abundant proteins bands that are characteristic of late Lyme disease are more predominant. I would be very skeptical of a Western blot that is positive for only 41 kDa, 31 kDa, and 34 kDa. That doesn't makes sense, certainly not for someone with Lyme disease. Hope this has been helpful.

P.S. All of this was known about OspA before the LYMErx vaccine was developed and used. In other words, vaccination was not considered to present a risk with regard to the laboratory diagnosis of Lyme disease since OspA did not seem to be all that relevant for diagnostic purposes anyway.

RitaA
Posts: 2768
Joined: Thu 1 Jul 2010 8:33

Re: Lyme neuroborreliosis test improvements

Post by RitaA » Tue 28 Aug 2012 0:50

Henry,

Thanks so much for your detailed response. It is very helpful to me in understanding why a positive band 31 and/or 34 -- even when accompanied by a positive band 41 -- might be viewed as a negative test result for Lyme disease.

I'm pretty sure I've read about 31kDa protein (OspA) being expressed mostly in the midgut of ticks before now, but it didn't register when I was reading your post, so I thank you for the reminder. If I'm understanding you correctly, it's theoretically possible to detect OspA in a human during a flare-up of Lyme arthritis, but there would likely be other bands (in addition to 41) showing up as well if the person had an active infection. I'm assuming that the flare-up you're describing would be more of an autoimmune response -- otherwise more bands (and especially 39kDa) would likely also be present.

I'm guessing the vaccine developers had to target something that would be detectable via testing in order for the vaccine to work. It sounds like they did their best to lessen any impact on blood test results.

Henry
Posts: 1108
Joined: Thu 10 Nov 2011 18:49

Re: Lyme neuroborreliosis test improvements

Post by Henry » Tue 28 Aug 2012 13:56

RitaA: Note that the LYMErx vaccine was designed to be a transmission-blocking vaccine. Humans immunized with LYMErx, which is an OspA-based vaccine, will make antibodies vs OspA as expected. When infected ticks take a blood meal from an immunized person, the OspA antibodies present will kill/neutralize Borrelia in the midgut of the tick and thereby negate their ability to transmit infection. Obviously, the presence of antibodies vs OspA will not induce protective immunity in vaccinated people because Borrelia make little or no OspA in the mammalian host. I'm amazed at how little those who criticized the LYMErx vaccine understood about how it was designed to work.

Lorima
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Re: Lyme neuroborreliosis test improvements

Post by Lorima » Tue 28 Aug 2012 19:48

This test from "Gold Standard Diagnostics" is a copy of the old test, except it uses "cloned or purified" proteins sprayed onto the membrane, instead of sonicated (broken-up) spirochetes separated on a gel and transferred onto the membrane. The same "bands" are said to be relevent, and the same interpretation criteria are used, as in the test successfully promoted and adopted in 1994, which is still being used today.

The ELISA is still "required" as a first step, so any cases missed by the ELISA will still be missed after the introduction of this test.

So it won't identify a greater percentage of actual Lyme disease patients, than the old test. But it will be marketed as a new and improved test that "fixes" the problems with the old test.

I know this is fine with you, Henry, as you believe the current test is adequately sensitive. Anybody else have an opinion? Mine is that the current test is too insensitive (that is, misses diagnosing too many actual LD cases) to be acceptable.
Last edited by Lorima on Tue 4 Sep 2012 22:15, edited 2 times in total.
"I have to understand the world, you see."
Richard Feynman

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

Re: Lyme neuroborreliosis test improvements

Post by radicale » Tue 28 Aug 2012 20:43

The performance of the tests is not acceptable. The reason is simple. Each region has it's own strains and in a global world like today where I can be in Europe, Japan and North America within a time-frame of a few weeks how would you know which strain I have been infected with. If the North American CDC guidelines were used with a test based on a North American strain it could very likely result in a false negative finding.
http://jcm.asm.org/content/35/6/1433.full.pdf
Interpretation criteria for standardized Western blots for three European species of Borrelia burgdorferi sensu lato.
Have a look at Fig. 4 showing the distribution of positive bands with respect to Disease presentation for the most prevalent strain in Europe. Even for arthritis only 80% of patients have 5 or more bands. For Neuroborreliosis only 20% have 5 or more bands. The authors go on to recommend a positive result if two or more specific bands are found.
http://www.ncbi.nlm.nih.gov/pubmed/19240047
Local Borrelia burgdorferi sensu stricto and Borrelia afzelii strains in a single mixed antigen improves western blot sensitivity.
The mixed antigen and revised interpretation criteria have been successfully incorporated into the routine diagnostic testing service, increasing the sensitivity of the in-house IgG western blot test for Scottish patients.
The above study shows that the use of mixed-antigen western blots can improve sensitivity and specificity significantly and truly illustrates the problem at hand.

http://www.provlab.ab.ca/LabBulletin201 ... isease.pdf
The potential diagnostic problems arising from strain diversity have been recognized in Alberta where a new ELISA is being used a screening test since March 2012. It only took them 20 years to figure out that most of the cases will be acquired out-of-province and as such a test that can detect these cases needs to be used.

Furthermore, what benefit does the current 2-tier test provide? During the early stage it's sensitivity is low and diagnosis is based on objective findings (tick bite, EM, flu) and during the late stage it's specificity is not good enough and once again is based on objective findings (arthritis)!? The test is clearly not good enough.

Edited to add:
http://www.ncbi.nlm.nih.gov/pmc/article ... 341732.pdf

The study we have presented has shown that the choice of particular B. burgdorferi strains for preparation of Lyme borreliosis Western blots does affect the level of reactivity detected with specific European sera.

In no cases would the use of B. burgdorferi sensu stricto strains offer performance advantages over the use of B. garinii or B. afzelii strains for testing the European sera, and in several cases, significantly lower levels of reactivity were observed.
A look at Table 2 reveals the importance of strain diversity. Another interesting study on ELISA sensitivities.
http://jcm.asm.org/content/36/2/427.full
mpact of Strain Heterogeneity on Lyme Disease Serology in Europe: Comparison of Enzyme-Linked Immunosorbent Assays Using Different Species of Borrelia burgdorferi Sensu Lato
A look at Table 2, once again, shows the importance of strain diversity and also illustrates the fact that the ELISA is not 100% sensitive...

http://jcm.asm.org/content/36/2/427/T2.expansion.html

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