Lyme disease (LD), caused by Borrelia burgdorferi
and transmitted by the bite of infected Ixodes
ticks, is the most common vector-borne disease in the United States (1
), with an estimated incidence of ∼300,000 cases per year (2
). Lyme disease typically begins with erythema migrans (EM), an expanding skin lesion at the site of the tick bite. If left untreated, spirochetes may disseminate from the site and patients may present with neurologic, cardiac, and/or rheumatologic manifestations (4
For the laboratory support of Lyme disease diagnosis, the Centers for Disease Control and Prevention (CDC) recommends a standard 2-tiered (STT) approach comprised of a first-tier enzyme immunoassay (EIA) that, if positive, should be followed by a second-tier IgM/IgG immunoblot assay (5
). The immunoblot assay is interpreted using standardized criteria, and the IgM immunoblot assay results are used only for disease of ≤30 days’ duration. While the STT approach has worked relatively well when used as recommended, there is plenty of room for improvement. The STT approach requires a complex laboratory infrastructure to perform and has a low sensitivity during early infection, inter- and intralaboratory variability, a long turnaround time, and a high cost because of the high cost for the immunoblot assay. There is also confusion regarding interpretation of the immunoblot assay results (5
). Over the last few decades, specific B. burgdorferi
epitopes have been mapped. Because only a yes-or-no result is needed for routine cases of suspected Lyme disease, hope has been raised that the STT approach can ultimately be replaced by a single test without the immunoblot assay.
Assays that improve upon the performance of current tests would be most helpful for the laboratory support of Lyme disease diagnosis. While next-generation diagnostic tests are suggested to be at hand (5–7
), there remains a need to demonstrate that known epitopes can adequately match the sensitivity and specificity of STT or whether further comprehensive exploration of epitopes is required. Most importantly, it has not been demonstrated that an effective single serodiagnostic test could be offered at the point of care. Rapid assays and point-of-care diagnostic testing could be used in some clinical settings, such as emergency rooms in areas of endemicity and doctors’ private practices (5
). Previously, we established a proof of principle for a new rapid test, the mChip-Ld assay, which was developed for point-of-care use (9
). Here, we report on the performance of an improved mChip-Ld assay using panels of serum samples from patients rigorously characterized to have confirmed early Lyme disease or Lyme arthritis (a late Lyme disease manifestation) and control serum samples from healthy individuals and individuals with look-alike diseases.
The development of assays for the laboratory diagnosis of early Lyme disease remains a challenging unmet need. We improved our microfluidics assay (mChip-Ld) for the rapid detection of B. burgdorferi antibody in serum from Lyme disease patients and analyzed its diagnostic performance against that achieved with samples acquired from three different sources: LDB, NIH, and CDC. The rapid mChip-Ld assay detected early Lyme disease in samples from patients with early Lyme disease with a higher sensitivity and a higher specificity than the STT algorithm.
Currently, CDC recommends the STT algorithm, a two-tiered testing approach comprised of a sensitive first-tier EIA that, if positive, is followed by a second-tier IgM/IgG immunoblot assay if the disease has been present for <30 days or an IgG-only immunoblot assay if the disease has been present for >30 days (25
). CDC has updated its recommendations for the serodiagnosis of Lyme disease by deeming those assays that use a second EIA in lieu of the immunoblot assay to be an acceptable alternative for the second tier of the STT algorithm (26
). Quantifiable EIA-based methods can provide objective test results, in contrast to the operator-dependent subjective interpretation of immunoblot assay results (5
Our antigen discovery was done by an EIA screen of old and new B. burgdorferi
diagnostic candidates against four panels of serum: three panels consisting of serum from patients in which early and late Lyme disease were confirmed (the LDB and NIH panels) or suspected (the NYSDOH panel) with a positive 2-tiered serology and one panel consisting of serum from negative controls (n
= 139 samples) (Table 1
and Fig. 1
), providing further evidence that several known antigens (5
) can be used to develop sensitive serologic assays for early and early disseminated/late Lyme disease. Our data also show that more antigens become positive with the progression of Lyme disease from the early to the late stage, from VlsE, OspC-K, and PepVF in the early acute phase to p100, ErpB, BmpA, VlsE, p28, DbpA, DbpB, and PepVF in Lyme arthritis. Thus, our data demonstrate the progression of the antibody response to specific antigens of B. burgdorferi
per disease stage, which is also seen in immunoblot assays (3 positive bands for IgM, >5 positive bands for IgG). Interestingly, for PTLDS, a protein usually associated with early infection, OspC (27
), detected PTLDS in 65% of the samples in the panel of samples from patients with PTLDS. Another interesting observation is that ErpB, which is associated with early disseminated and late Lyme disease (16
), detected positive samples in the panel of samples from patients with PTLDS with a 50% sensitivity. The importance of these findings requires further study.
Our screening study identified three markers that performed with a considerably high sensitivity for the detection of anti-B. burgdorferi
antibody in serum from patients with early Lyme disease: VlsE (79% and 95%), OspC-K (47% and 75%), and PepVF (74% and 95%). Two of these markers (OspC-K and PepVF) were previously identified in our proof-of-principle study in which we used a small (n
= 35) but rigorously characterized panel of serum samples from patients with Lyme disease from the CDC that mostly included samples from patients with convalescent-phase and late Lyme disease (9
). Interestingly, subsequent to the proposed use of the PepVF sequence (14
), independent large-scale screening efforts identified the same VlsE (29
) and FlaB (29
) epitopes contained in PepVF. The observed sensitivity of the VlsE antigen for the detection of all cases of Lyme disease may be explained by the association of an immune response toward specific VlsE sequences during early and late stages of the disease (31
). Furthermore, anti-VlsE antibody was identified in the anti-Borrelia burgdorferi
profiles of PTLDS patients (32
), which is also supported by the findings of our study.
Microfluidics offers practical advantages for miniaturizing laboratory-based tests, including portability, multiplexing, speed, and performance (19
). Overall, the performance of the mChip-Ld platform largely matched the performance of our laboratory-based 3Ag-EIA IgG functionalized with the same antigens. The improved performance seen in some cases (e.g., samples from patients with early-stage disease, especially those in the CDC and LDB panels) could be due to the mChip-Ld platform detecting IgM antibodies, which peak in the first 2 to 6 weeks after disease onset (34
), in addition to IgG, which was the only isotype detected in our EIAs. Traditionally, IgM detection decreases the specificity of an assay (35
) and an increase in sensitivity is counterbalanced by a decrease in specificity. Avoiding low specificity was the reason for our exclusion of IgM detection in our antigen discovery phase using EIA. Our IgM/IgG mChip-Ld results showed sensitivities of over 80% to 100% for the panels of serum samples from patients with Lyme disease tested and specificities of 100% with an AUC of 0.865 to 0.941 for the two panels with both positive and negative specimens. These data show that in the microfluidics platform, an increase in sensitivity was not followed by a decrease in specificity, as we predicted for the combined IgM/IgG detection. One possible explanation is that the optimization of parameters specific to an assay and antigen molecules (36
) can significantly alter the performance of the microfluidics assay. Here, additional improvement of assay conditions, particularly in reducing the variability of the internal positive- and negative-control signals through buffer modification, was carried out prior to the testing of the specimen panels. Furthermore, for the multiplexed testing, while we previously summed the three OD signals with equal weights (9
), here, our final score consisted of a sum of weighted quantitative measurements which were determined empirically (23
). Such improvements in assay technology and the multiplexed algorithm contributed to an increased overall sensitivity and specificity of the mChip-Ld. However, further testing in a clinical setting is necessary to confirm whether the mChip-Ld format can consistently perform better than the 3Ag-EIA.
There is no commercially available rapid point-of-care diagnostic test for Lyme disease (19
). This work demonstrates an approach that could lead to an objective, point-of-care test for Lyme disease (9
) with a diagnostic performance that matches that of current standard laboratory testing or the, in some cases, outperforms current standard laboratory testing with the potential to be a stand-alone replacement for the STT algorithm. The mChip-Ld performed with a sensitivity either similar to or higher than that of the STT algorithm without losing specificity, which remained above 95%. More broadly, this study demonstrates the potential of the microfluidics technology to deliver high performance for multiplexed assays in a portable format in an era when immunodominant epitopes are increasingly being identified for a wide array of infectious organisms.
The cassettes and reagents are from Opko; all reasonable requests for materials sharing will be considered.
We thank Martin Schriefer from the National Center for Infectious Diseases, Centers for Disease Control and Prevention, for providing the Lyme disease-characterized serum panels (CDC Lyme disease panel) and Christopher Sexton for revealing the contents of the panel after the data were acquired.
This work was supported by Public Health Service grant R44 AI096551 to M.G.S. via Immuno Technologies Inc. and in part by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
The content of this publication does not necessarily reflect the views or policies of the U.S. Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the U.S. government. The funders for the study provided support in the form of salaries for authors but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The corresponding authors had full access to all the data in the study and had final responsibility for the decision to submit the manuscript for publication.
M.G.-S. is an employee of Immuno Technologies Inc. and holds a 5% or greater financial interest in Immuno Technologies Inc. V.L. was an employee of Opko Diagnostics LLC while engaged in the research project. M.G.-S. and A.R.M. hold relevant patents. V.L. declares a financial interest in Opko Diagnostics. S.A., S.N., F.S.D.S.M., T.W., R.C.C., M.S.G., S.J.W., and S.K.S. declare no competing financial interests.
S.A., S.N., M.G.-S., and S.K.S. designed the study; S.A., F.S.D.S.M., and R.C.C. performed the microfluidic immunoassays; T.W. and M.S.G. performed the EIA and protein purification; V.L. advised on assay development and provided materials and reagents; S.A., S.N., T.W., M.G.-S., and S.K.S. analyzed the data; and S.A., S.N., M.G.-S, A.R.M., and S.K.S. wrote the paper. E.J.H., S.J.W., and A.R.M. provided Lyme disease-characterized serum panels. All coauthors edited the paper. All figures and tables were created by an author of the paper.