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American Lyme Disease Foundation, Inc.

American Lyme Disease Foundation, Inc.
P.O. Box 466
Lyme, CT 06371

The Laboratory Diagnosis of Lyme Disease II
The Banbury Center, Cold Spring Harbor
9-12 September 2007
Emerging Views on Serodiagnosis

Serology is at present the most useful tool for assessing exposure to B. burgdorferi in all stages of Lyme disease after the first weeks of infection. This conference focused on serology in view of its key role in supporting the clinical diagnosis of Lyme disease and because of significant new developments in the field. An array of possible alternatives or improvements to the current two-tier test system for the serodiagnosis of Lyme disease was discussed. Among the various possibilities were peptide based assays, improved data analysis, and the use of new target antigens identified through proteomic studies. It was generally felt that none of the currently available methods are ideal and that additional research and development are needed to produce better sero- and non-serodiagnostic assays. At the conclusion of the Banbury conference, attendees discussed: 1) the use of a commercial C6 peptide enzyme immunoassay as a “stand alone” serologic test for the laboratory diagnosis of Lyme disease in the United States or as a replacement of IgM assays for the diagnosis of early infection and 2) the need for a serum repository as a resource for research and test evaluation.

In 1995, the U.S. Centers for Disease Control and Prevention recommended a two-tiered scheme for testing serum samples for antibodies to Borrelia burgdorferi sensu stricto as an initial step to standardize serology. This recommendation was published (MMWR 1995) after review of the state of the field by representatives of diverse interests including academic reference laboratories, state health departments, test developers and vendors, clinical laboratory standards organizations, and agencies of the United States Public Health Service (Second National Conference on Serologic Diagnosis of Lyme Disease, Dearborn, MI; October 27-29, 1994). The Dearborn conferees recognized that new serologic methods may improve capabilities to evaluate patients for Lyme disease. These methods could replace one or both tests of the recommended two-tiered protocol if their performance (specificity, sensitivity, and reproducibility) was determined to be equal to or better than the performance of the two-test procedures.

The Banbury conferees were in agreement that the two-tiered system, particularly the measurement of IgG antibodies, has generally worked well. Two-tiered detection of IgG antibodies to Borrelia burgdorferi is highly specific and sensitive. However, in the early weeks of infection, two-tiered serology has low sensitivity and is not recommended for evaluation of patients with erythema migrans. The specificity of two-tiered testing for IgM antibodies is less than ideal; in consequence, IgM testing should be performed only in the first month of illness. In view of the complexity of two-tiered testing and the shortcomings of current IgM serology, there was broad interest in alternative tests. Some even felt that two-tiered IgM testing using current methods should not be done, especially since good clinical information about the duration of illness is often lacking in requests for testing. The inappropriate use of serology to test patients with a low pretest probability of Lyme disease was recognized as an ongoing problem. The pretest probability of disease has a very important impact on test interpretation; this concept should be emphasized in educational efforts in order to improve care of patients.

The most highly developed and evaluated alternative to two-tiered serology is a C6 EIA. This assay is based on a peptide (C6), whose amino acid sequence reproduces a conserved, immunodominant region of a single protein of Borrelia burgdorferi. There is an extensive peer-reviewed literature on the performance of the C6 peptide as antigen in various assays and an application to have the commercial C6 assay approved for use as a “stand alone” test has been submitted to the Food and Drug Administration. If the C6 assay were to be cleared by the FDA for this purpose, physicians could choose either the C6 test or two-tiered procedures for evaluating patients. In research settings, this single antigen assay could serve as a simpler, less costly, and more objective standard against which the performance of new technologies could be judged.

Unpublished data comparing the performance of the commercial C6 assay with two-tiered testing were presented at the meeting. The principal findings reported were that the sensitivity of the C6 assay was significantly greater than two-tiered testing (p<0.05) in early infection, defined as up until 21 days after the onset of erythema migrans. After this time, the sensitivities of the two types of testing were not statistically significantly different. The C6 assay was positive in EM patients infected with a broader range of B. burgdorferi genotypes than was two-tiered testing using B. burgdorferi sensu stricto antigens. Specificities of the C6 assay and two-tiered testing in a population of healthy donors from regions of the U.S. non-endemic for Lyme disease were both above 99%. The specificity of two-tiered testing in the non-endemic population exceeded that of the C6 EIA by 0.6%, a result that was not significantly different (p=0.37). Among donors from endemic areas, the specificities of both types of testing were approximately 0.5% lower (98.6 vs 99.4), but also not significantly different. Among the entire blood donor population (n=1842), two-tiered testing yielded a significantly higher specificity of 99.4% compared with 98.6% for the C6 EIA (p=0.03). Testing of 366 serum samples from individuals with one of 14 other diseases or with one of several interfering conditions yielded a higher specificity for the C6 ELISA of 99.5% compared with 99.2% for two-tiered testing, but this difference was not significant.

In the discussion of these data, other groups using C6 assays that they had developed in their own laboratories raised questions about the specificity of C6-based assays. Two groups noted that C6 sequence variation among the various genospecies of Borrelia burgdorferi sensu lato results in differences in their ability to detect antibodies, but these differences were not described as significant.

Assessment of the appropriate role of a “stand alone” C6 assay must await publication of the complete data set. Conferees offered their emerging views based on the information presented. Some physicians and public health scientists were impressed particularly with the apparent ability of the C6 assay to improve early detection of antibodies and with its simplicity and objective score. Others felt that C6 still does not provide a sufficient degree of specificity to warrant its use as a stand alone assay, especially in late disease.

Views were mixed about the use of the C6 assay as a stand alone test in late Lyme disease (arthritis, carditis, or neuroborreliosis). Some saw the lack of a statistically significant difference in specificity between the commercial C6 test and two-tiered testing in the reported study as evidence of the non-inferiority of the C6 assay. They looked to FDA for a definition of “non-inferiority” to guide the field. An alternative view was that specificity is paramount in late Lyme disease and so any new assay should be equal or superior to the specificity of the two-tiered system. The slightly lower specificity observed for the commercial C6 assay was considered worrisome, as well as the fact that the test depends on a single antigen and a single commercial supplier. IgG immunoblots reveal the complexity of the immune response in later disease, which can be diagnostically useful.

A proposal was presented to replace two-tiered IgM serology with the C6 EIA as a stand alone test in early disease and actively discussed. The proponents of the commercial C6 assay noted that although the C6 assay detects mainly IgG, it becomes positive earlier in infection than standard IgM serology. Nevertheless, a negative serologic result in early infection does not rule out Lyme disease. Indeed, no serologic test may rule out infection if a blood sample is taken so early that an individual has not had time to develop antibodies to any antigen. It may be possible to further improve serology of early disease by identifying other antigens, for example, by proteomic studies. However, as with the whole-cell antigen ELISA, specificity may diminish as more antigens are included. Data were presented that suggested that two additional antigens may augment the sensitivity of the C6 IgG assay, without compromising specificity, but prospective studies using larger data sets are needed to confirm these preliminary results.

For patients with clinical manifestations of Lyme disease other than erythema migrans, C6 was discussed as a first-tier test, a use that has been cleared by the FDA for the current version of the commercial assay. A C6 result in the positive or indeterminate range requires a positive IgG immunoblot in order for the sample to be scored as positive. Some argued that blotting is needed to avoid false positive results in patients evaluated for late Lyme disease. Anecdotal evidence of false-positive C6 tests in various ophthalmic conditions (optic neuritis, uveitis, and a few multiple sclerosis patients) was reported. Others thought that the data presented indicate that the commercial C6 assay (slightly modified from the version cleared by the FDA) is sufficiently specific to make blotting unnecessary.

An alternative proposal was to combine whole cell sonicate ELISA as a first-tier test with IgG immunoblot that includes a VlsE stripe as the second-tier assay. This is based on the observation that Borrelia burgdorferi whole cell lysate ELISA is more sensitive than the C6 EIA, albeit much less specific. In Europe, sensitivity of the IgG whole cell extract ELISA improved further when rVlsE was added. The reduced specificities of whole cell ELISAs could be obviated by a second tier test consisting of an IgG blot with a VlsE stripe. Scoring criteria for such a blot were proposed but have not yet been validated.

European participants voiced concern about relying on a single test antigen in Europe, where multiple species of Borrelia cause Lyme disease. Reference was made to a Quick ELISA C6, which may not be as sensitive as the commercial test approved for use in the United States and now marketed in Europe. Immunoblots and line assays with selected recombinant antigens improved sensitivity in European patients with early disease, especially early neuroborreliosis. Specificity was improved when two or more antigens were required to be reactive in blot formats. To date, a direct comparison of the performance of the current commercial C6 peptide ELISA with multi-antigen blot tests with a sufficiently large number of samples has not been made.

Proteomic approaches are being used to discover new antigen candidates for potential recombinant or peptide-based multi-antigen tests. In principle, the products of all open reading frames can be evaluated in microarray format, including antigens that are only expressed in mammalian hosts. Work is in progress to determine whether newly discovered antigens may outperform the well-studied tests.

There was a strong consensus that a new national serum repository should be developed. Without such a collection of serum samples it will be difficult to develop and evaluate new tests and to compare one test with another. A committee was formed to determine the number, types, and quantities of samples to be obtained. CDC pledged $100,000 (subsequently increased to $200,000) in contracts toward this effort in fiscal year 2008 and offered to manage the specimen collection. Supplementary funds for this purpose will be sought from diverse sources (NIH, FDA, and SBIR grants) in the coming months.