The evolution of Borrelia serology tests.
The first protocol
In 1994, several organizations and agencies, including the Association of State and Territorial Public Health Laboratory Directors (now known as the Association of Public Health Laboratories [APHL]), the Centers for Disease Control (CDC), the National Institutes of Health (NIH), and the U.S. Food and Drug Administration (FDA), held a meeting in Dearborn, Michigan: the Second National Conference on Serologic Diagnosis of Lyme Disease. (1) Recommendations that emerged from that meeting resulted in guidelines for the serological protocol to aid in the diagnosis of LD as well as recommendations for interpretation of these serology tests. The outcome, the "Two-Tier Test Protocol," is still the standard in North America.
Briefly, this protocol involves first screening serum specimens on a sensitive immunoassay designed to measure IgG and IgM antibody to B. burgdorferi (typically an enzyme-linked immunosorbent assay [ELISA] or indirect fluorescent assay [IFA]). Since this first tier of the testing protocol serves as an initial screen, it is imperative that such assays are superbly sensitive, even at the possible expense of ideal specificity. As the Clinical and Laboratory Standards Institute (CLSI) points out in its guidance document, (2) a negative screening result should indicate that the person has a high probability of being free of the disease, whereas a positive test result might reflect only the need for further testing.
Specimens positive or borderline on the screening assay are to be further tested by separate IgG and IgM immunoblots. Specimens negative on the initial screen are to be reported as negative. Specimens positive or borderline on the initial screen and found to be negative on both the IgG and IgM immunoblots are to also be reported as negative to Borrelia burgdorferi antibody. Those found to be positive by screen and IgG positive by immunoblot should be reported as positive, and those positive by screen and IgM positive by immunoblot should be reported as positive; however, that positive IgM result is only clinically significant in very early disease.
As is the case with many other infectious disease immunoassays, serology tests designed to measure antibody to Borrelia have changed by design through the years. Through my last query of the FDA 510(k) database, I have uncovered a total of eighty-two products cleared by the FDA. The first, a Borrelia burgdorferi IFA test system, was cleared in 1987 and is still in use. While this test system has been used in clinical laboratories for more than 27 years, it is not without limitations. A highly skilled and well-trained technician is required to perform the assay and interpret the results. The results are somewhat subjective because the assay requires a visual interpretation.
In 1988, just months after the commercialization of the IFA test, the first commercial ELISA tests for antibody to B. burgdorferi were introduced into clinical laboratories. Like the IFA, these ELISA tests incorporated whole cell B. burgdorferi as the solid phase. Unlike the IFA, the early ELISA tests provided an objective result and could be easily automated on open robotic analyzers. While some of these diagnostic manufacturers have disappeared from the market, some have not, and whole cell Borrelia ELISA tests are still widely used today.
As researchers learned more and more about the nature of Borrelia antigen expression, and with the availability of recombinant and synthetic B. burgdorferi proteins, it was apparent that Borrelia serology was transitioning. In 2001, the first ELISA test utilizing a single, synthetic antigen as the antibody capture phase was cleared by the FDA. That test was the C6 ELISA, and it is also still in use. This assay utilizes a 26 mer synthetic peptide (the C6 peptide) based on the invariable region 6 of the VlsE (Vmp-like sequence, expressed) lipoprotein of B. burgdorferi. (3)
In 2003, Bacon and others (4) conducted an extensive serological analysis of 280 diverse cases of LD patient samples as well as 559 non-LD control specimens. They investigated IgG and IgM antibody activity to recombinant (r) VIsEl, synthetic C6, and synthetic pepCIO (a 10-amino acid, conserved synthetic peptide found at the C terminus of the Borrelia OspC [outer surface protein C] protein). OspC is a major surface lipoprotein of B. burgdorferi (5) and is one of the three Borrelia proteins key in interpreting IgM Western blots. ELISA results utilizing the purified antigens (recombinant or synthetic) were compared to the two-tier method utilizing whole cell antigen. The study showed that the combination of rVlsEl IgG (or C6 IgG) and pepC10 IgM maintained high specificity (98%) and was more sensitive than the two-tier method in patients with acute LD. In later cases of LD they were statistically equivalent. Additionally, the combination of rVlsE1 IgG (or C6 IgG) in conjunction with pepC10 IgM was superior to rVlsel, C6 or pepC10 alone.
As a result of the Bacon study, a new multiplex immunoassay (6) as well as an ELISA test were developed and manufactured that utilized rVlsE1 and synthetic pepC10 (dual antigens) as the solid phase. Cleared by FDA in 2010 and 2012 respectively as first-tier Borrelia screening assays, these test systems also have no whole cell antigen on the solid phase.
Borrelia serology has evolved quite a bit over the past decades. Many commercial immunoassays have been developed incorporating whole-cell antigens, and some feature highly specific and sensitive recombinant and synthetic Borrelia peptides. Both types are in widespread use today. What types of assays are used by individual laboratories and/or associated infectious disease physicians therefore seems to be a matter of preference. In fact, Ang and others (7) highlight potential performance differences among Borrelia tier-one tests, tier-two immunoblots, and especially the combination of pairing them together. They demonstrate that the selection of that combination can result in widely divergent sensitivity and specificity performance, and that is independent of the nature of the antigens used in the various immunoassays. Clearly, as additional sensitive and specific Borrelia proteins become available, it is almost certain that the serology assays will continue to evolve in an effort to improve their clinical performance.
(1.) ASTPHLD and CDC. Proceedings of the second national conference on serologic diagnosis of Lyme disease, Dearborn, Ml, 27-29 October 1994.
(2.) CLSI. Specifications for immunological testing for infectious diseases; Approved Guideline--Second Edition. CLSI document I/LA18-A2 [ISBN 1-56238445-7], CLSI, Wayne, Pennsylvania 19087-1898 USA, 2001.
(3.) Marques A, Martin D, Philipp M. Evaluation of the C6 peptide enzyme-linked immunosorbent assay www.mlo-online.com for individuals vaccinated with the recombinant OspA vaccine. J. Clin. Microbiol. 2002;40:2591-2593.
(4.) Bacon R, Biggerstaff BJ, Schriefer ME, et al. Serodiagnosis of Lyme disease by kinetic enzyme-linked immunosorbent assay using recombinant VlsE1 or peptide antigens of Borrelia burgdorferi compared with 2-tiered testing using whole-cell lysate. J Infect Dis. 2003;187:1187-1199.
(5.) Utpal Pal, Xiaofeng Yang, Manchuan Chen, et al. OspC facilitates Borrelia burgdorferi invasion of ixodes scapularis salivary glands. J. Clin. Invest. 2004;113(2):220-230.
(6.) Porwancher R, Hagerty CG, Fan J, et al. Multiplex immunoassay for Lyme disease using VIse1-IgG and pepC10-lgM antibodies; improving test performance through bioinformatics. Clin. Vaccine Immunol. 2011;18(5):851-859.
(7.) Ang CW, Notermans DW, Hommes M, et al. Large differences between test strategies for the detection of anti-Borrelia antibodies are revealed by comparing eight ELISAs and five immunoblots. Eur J Clin Microbiol Invect Dis. 2011;30:1027-1032.
Mark Kopnitsky serves as Vice President of Science and Quality for ZEUS Scientific. ZEUS has cleared eight different types of Borrelia immunoassays through FDA, and develops/produces FDA-cleared Borrelia immunoassays in virtually all formats including IFA, ELISA, Western blot, and the multiplex AtheNA Multi-Lyte format. ZEUS produces and markets FDA-cleared ELISA tests using whole cell antigen as well as ELISA tests using recombinant and synthetic antigens.
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|Author:||Kopnitsky, Mark J.|
|Publication:||Medical Laboratory Observer|
|Date:||Jun 1, 2014|
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