Lyme Disease: Rolling the dice with ELISA

4. June 2013
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In the clinic and the laboratory a war of a special kind is brewing. Ticks transmit Borrelia burgdorferi to 200,000 people each year, year in year out; a vaccine does not exist. Tests have rather big shortcomings – and so some infection remains undetected for years.

Lime Green Light Games: On 10 and 11 May Niagara Falls and the CN Tower were made to shine in unaccustomed splendour; similar actions are also planned for Berlin. This is no mere art performance for art’s sake – rather, patients worldwide are trying to bring the attention of doctors and politicians to lyme disease. This activism may seem surprising, but is being played out against a serious backdrop. Timely detection of uncomplicated infections can be treated well with antibiotics. Without adequate treatment, fatigue often occurs, as do joint and muscle pain, and cognitive deficits. In late stages the central and peripheral nervous system are affected (Neuroborreliosis).

Myths and Misconceptions

Following a tick bite the disease begins with local infection, including rash (Erythema migrans), ‘flu-like and sometimes gastrointestinal symptoms. John J. Halperin, Phillip Baker and Gary P. Wormser as part of a review article criticised the fact that doctors tend to base too much of their diagnosis on the respective characterics on the skin. The fact is that up to 50 percent of patients do not develop redness. Antibodies do show up in the blood – but this can take several weeks. If, after the end of therapy, the telltale signs of erythema migrans recur, the fear arises that patients have relapsed. Now scientists have shown by using genetic analysis that  –  in a process completely separate from the ailment already undergoing treatment – a new infection of Borrelia is the cause.

Much Trouble with Antibodies

In suspicious cases specialised laboratories will analyse a blood sample using enzyme-linked immunosorbent assays (ELISA) for antibodies. The method has several drawbacks: sensitivity, specificity and standardisation do not meet expectations. According to Dr. Armin Schwarzbach from the Borreliose Centrum Augsburg up to 70 percent of all ELISA tests turn out negative despite presence of infection. “The problem is that in the ELISA tests too few recombinant antigens and lysates are used in what are either wrong or incomplete blends. We now know that there are numerous new Borrelia subspecies”, says Schwarzenbach. The pronounced antigenic variability and variation between individual subspecies is problematic. In Russia, Japan, Australia and Europe various different detection methods would be required. Currently, there are about 30 different ELISAs on the market – using different antigens.

Standards? Forget it!

It’s true that the Medical Devices Act prescribes no standardisation of tests. However, laboratories should participate in successful proficiency testing in order to conform to external quality criteria. Schwarzenbach: “In my eyes, standardisation ought to be the responsibility of the German National Reference Centre (NRZ) for Borrelia. For seven years, nothing’s happened”. Both the antigens and the reference areas should be standardised. “They can’t just pull out one reference field and use it for all of the stages of disease. In the individual stages there are completely different antibody concentrations in serum and in cerebrospinal fluid”, Schwarzenbach points out. “Routine laboratories cannot afford it, they adopt the reference field from the test manufacturer. Physicians working in the clinic who issue the examinations often do not know this”. Depending on the publication, the sensitivity of the assay is stated as being at 30 to 60 percent. Only after a positive or at least borderline ELISA result is the finding permitted to be checked via immunoblot, otherwise health insurance funds do not cover costs. “Immunoblots are indeed more sensitive than ELISA tests but do not exclude possible presence of infection. The GKV (association of obligatory health insurance providers) however only refunds for the “dice game” being played with ELISA and therefore leaves the fate of the patient in the hands of the manufacturers employed”,  the researcher criticises. Immunoblots themselves achieve only 60 percent sensitivity. “The last attempt to carry out a ring trial for neuroborreliosis on cerebrospinal fluid was quite a disaster, results can vary by a factor of eight“, says Schwarzenbach.

Response from the NRZ

Upon request by DocCheck a spokeswoman for the NRZ reported about current work on the standardisation of lyme disease diagnostics: their hospital is currently working on improving their blots based on recombinant antigens. In addition the development, testing and introduction of evidence based on VIsE (variable major protein-like sequence expressed), as well as the testing of and implementation of an online test format, will enter the picture. This is a system based on IgG Western blots. Further points of emphasis mentioned by the spokeswoman include multiplex assays, as well as advice on the development and validation of tests of various formats. She adds that the NRZ also makes materials – in particular recombinant antigens monoclonal antibodies, patient sera, and Borrelia strains – available to companies. In addition, a serum panel will be established.

News from the Lab

In addition to ELISA and immunoblots, methodologically sophisticated lymphocyte transformation tests (LTT) have been established. Laboratories isolate lymphocytes from a blood sample and determining the extent to which a response to specific antigens then follows. “I think the test is helpful, but is equally neither exclusive nor conclusive”, says Schwarzenbach. There remains the possibility of tracing bacterial genome in aspirates, tissue samples or biopsies via a polymerase chain reaction (PCR). A negative result does not necessarily also mean that patients are Borrelia-free. With respect to neuroborreliosis, there now exist interesting projects which are studying CXCL13 as a marker. In early phases, the levels of this chemokine in the cerebrospinal fluid increase and then decrease with successful treatment. Schwarzenbach says the method could be useful as a supplement, since only seven to nine percent of patients with chronic neuroborreliosis had a positive antibody index . All in all, the situation is nevertheless extremely unsatisfactory. Therefore, the demands for evidence-based diagnosis are getting ever stronger.

The Long Road to a Guideline

At the moment, there exist only two S1 guidelines with the  Association of the Scientific Medical Societies in Germany , namely “Cutaneous Manifestations of Lyme Disease” and “Neuroborreliosis”. In addition, there is the guideline “Diagnosis and Treatment of Lyme Disease” from the German Borreliosis Society. Schwarzenbach: “Unfortunately, scientifically weak recommendations are always being misleadingly sold as guidelines. Our medical colleagues have believed that these correspond with the truth and had no shortcomings”. For this reason several specialist firms plan to submit a S3 guideline. Whether the paper will be free of any bias is questionable. The action alliance against tick-borne infections in Germany under Professor Sebastian Rauer, Freiburg, looks for potential conflicts of interest through its consultative and verification activities in the pharmaceutical and diagnostic industries. Rauer is one of the coordinators of the project. The next problem: “A few older studies exist – that’s it. Patient data might be there, but it first of all has to be evaluated”, says Schwarzenbach.

There is a lot to do

An objective S3 guideline on the diagnosis and treatment of lyme disease would  surely help our colleagues further. Schwarzenbach also makes demands for studies on antibody cut-offs at various stages of lyme disease. Technical articles often refer to outdated works from the 1990s. In addition, antibody tests and immunoblots ought to be “urgently standardised by the NRZ”, namely “taking into account the three stages, assuming that one accepts the third stage as given by the NRZ on lyme disease as a multi-system disease”.  Either way, ELISAs have technical limitations. “We need tests that also say something about the disease activity, antibodies are not up to the task”, says Schwarzenbach. In addition, the doctor calls for  “training, education and university studies, which accept that chronic lyme disease exists”. So long as no suitable evidence emerges, there remains solely the clinical, differential-diagnostic path.

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2 comments:

marketing
marketing

I am so grateful for your article post.Thanks Again. Great.

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MD Maria Edite Rio
MD Maria Edite Rio

Thank you for doing this alert. Also in Portugal we have the same problem with diagnose and the decision to treat. I have organized a conference in March 2009 at Oporto, about these dificulties in diagnose and treat, and some similarities with MS. We had the oportunity to have with us Prof Israel Steiner, but now everything has gone, and may be a good idea to organize another one.
Maia Edite Rio

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