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Journal of Clinical Microbiology, December 2002, p. 4782-4784, Vol. 40, No. 12
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.12.4782-4784.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Comparison of the Borrelia DotBlot G, MarDx, and VIDAS Enzyme Immunoassays for Detecting Immunoglobulin G Antibodies to Borrelia burgdorferi in Human Serum

Deborah J. Jespersen,¹ Thomas F. Smith,¹ Jon E. Rosenblatt,^1,2 and Franklin R. Cockerill III^1,2*

Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology,¹ Division of Infectious Diseases, Department of Internal Medicine, Mayo Clinic, Rochester, Minnesota 55905²

Received 1 May 2002/ Returned for modification 4 August 2002/ Accepted 13 September 2002

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Three enzyme immunoassays, Borrelia DotBlot G (GenBio, San Diego, Calif.), MarDx EIA (MarDx Diagnostics, Inc., Carlsbad, Calif.), and VIDAS (bioMérieux, St. Louis, Mo.) were compared for their ability to detect immunoglobulin G antibodies to Borrelia burgdorferi in 100 human serum samples. The "gold standard" positive result for each of these samples was determined by Western immunoblot analysis (MarDx Marblot Test System) (n = 99) or clinical signs and symptoms suggestive of Lyme disease with other laboratory results positive for B. burgdorferi (n = 1). Based on these criteria for a gold standard positive result, 29 of the 100 samples tested were considered true positives and 71 were considered true negatives. The following sensitivities and specificities were noted, respectively, for each method: Borrelia DotBlot G, 93 and 90%; MarDx, 100 and 35%; and VIDAS, 100 and 92%. Because of high sensitivity and specificity and ease of use, the VIDAS test is an appealing method, especially for laboratories that perform high volumes of tests. The high sensitivity but low specificity of the MarDx method compared to the VIDAS and Borrelia DotBlot G methods requires that Western blot confirmatory tests be performed frequently. The Borrelia DotBlot G method has acceptable specificity but appears to lack sensitivity when compared to the VIDAS and MarDx methods.

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Persons with disseminated or late-stage Lyme disease almost always have serological responses to Borrelia burgdorferi antigens. The current algorithm for Lyme disease serologic testing in the United States involves a two-tiered approach (5, 7-10). Sera are screened for immunoglobulin G (IgG) antibodies usually by enzyme immunoassay (EIA) or indirect immunofluorescence assay. Positive results for these assays are then confirmed by Western immunoblotting. Sera from patients that are negative by EIA are not tested further. However, if a patient is suspected to have early Lyme disease and has a negative EIA result, paired acute- and convalescent-phase serum samples are recommended.

Three commercial immunoassays have gained popularity as screening tests for the detection of IgG antibodies to B. burgdorferi in human serum, the Borrelia DotBlot G (GenBio, San Diego, Calif.), MarDx EIA (MarDx Diagnostics, Inc., Carlsbad, Calif.), and VIDAS (bioMérieux, St. Louis, Mo.). The Borrelia DotBlot G is a qualitative enzyme immunoassay for the presumptive detection of B. burgdorferi IgG antibodies in serum. For this assay, various antigens are incorporated in discrete dots on a nitrocellulose strip. The antigens distributed in these dots include whole-cell antigens, recombinant protein 93-kDa antigen, purified flagellin, recombinant P39 41-kDa antigen, recombinant OspC 23-kDa antigen, and a positive reagent control. These strips are dipped into a serum diluted in a reaction vessel and incubated. In the second stage, alkaline phosphatase-conjugated antihuman antibodies are reacted with bound patient antibodies. Finally, the strip is transferred to an enzyme substrate reagent, which reacts with bound alkaline phosphatase to produce a distinct dot. One potential limitation of the assay is that results are interpreted by the human eye. In our experience, interpretation of results may be difficult depending on the depth of purple color in the dot. Another limitation is that the technologist is nearly constantly dipping the strips, so that approximately only eight tests can be performed simultaneously.

The MarDx EIA (IgG) test is also an EIA-based technique. This method uses a mix of unspecified antigens extracted from B. burgdorferi (strain B 31) bound to polystyrene microwells. Serum is added to the microwells in the first step of this procedure, and antibody in the serum binds with the antigen. Following a rinsing step which removes unbound antibody, a peroxidase conjugate, a color indicator solution is added to the microwells which will react only in the presence of bound peroxidase. An acid solution is added after a specified period of time in order to stop the enzymatic conversion of the indicator solution for spectrophotometric analysis.

In contrast to the Borrelia DotBlot G and MarDx assays, the VIDAS test is an enzyme-linked fluorescence assay that uses fluorescence indicators instead of colorimetric indicators. VIDAS tests contain a solid phase receptacle (SPR) and a strip. The SPR is the solid phase of the reaction, which is disposable, and acts as a sampling needle that eliminates potential intersample contamination. The strip contains all the reagents which are ready for use. At each stage in the immunoanalysis, the SPR repeatedly takes up the reagent automatically and transfers it into the various wells of the strip, through to the final stage of the analysis. The last well is the reading well, where the final intensity of the reaction is measured by fluorescence.

The objective of the present study was to compare the abilities of these three enzyme immunoassays to detect IgG antibodies to B. burgdorferi in 100 human serum samples.

One hundred well-characterized serum samples (45 samples from a Centers for Disease Control and Prevention [CDC] Lyme disease proficiency panel and 55 from Mayo Clinic patients) were evaluated for the presence of antibodies to B. burgdorferi, using the Borrelia DotBlot G, MarDx, and VIDAS assays according to the manufacturers' instructions. The "gold standard" positive results for each of these samples were determined by either Western blot analysis (99 samples) or clinical signs and symptoms compatible with Lyme disease with a nonconfirmatory Western blot analysis but other laboratory tests positive for Lyme disease (1 sample). The Western blot method used was the MarDx B. burgdorferi (IgG) Marblot Test system (MarDx Diagnostics, Inc.). This assay was used according to the manufacturer's instructions, and a positive Western blotting result was defined according to CDC criteria (2; CDC presentation, Association of State and Territorial Public Health Directors, Washington, D.C., 1994). Based on these criteria for a gold standard positive result, 29 of 100 samples tested were considered true positives and 71 were considered true negatives.

Results are summarized in Tables 1 and 2. Among the three methods evaluated, the VIDAS test had the best performance (sensitivity, 100%; specificity, 92%). Although the sensitivity of the MarDx method (100%) equaled that of the VIDAS method, the MarDx assay had a considerably lower specificity (35%). The Borrelia DotBlot G method had a lower sensitivity (93%) than either of the other methods but a specificity (90%) that was similar to the VIDAS method's but much greater than the MarDx method's.

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TABLE 1. Results for three commercial EIA used to detect B. burgdorferi IgG antibodies in human serum

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TABLE 2. Sensitivities and specificities for three commercial EIA

The results of this study demonstrated that although the sensitivities for detecting B. burgdorferi IgG antibodies were nearly the same for three first-tier EIAs, the MarDx method was considerably less specific than the VIDAS or Borrelia DotBlot G methods. Therefore, more confirmatory Western blot assays would be required for the MarDx than with the Borrelia DotBlot G or VIDAS assays. This additional confirmatory testing adds to laboratory technologists' time and expense and also delays the time that the final result is available to the clinician. In the present study, 40 confirmatory Western blots would have been eliminated if the VIDAS immunoassay was performed instead of the MarDx EIA and 39 confirmatory Western blots would have been eliminated if the Borrelia Dot- Blot G immunoassay was performed instead of the MarDx EIA. The cost of performing a Western blot in our laboratory is approximately $20.00 per sample. The VIDAS is a fully automated test system suitable for high-volume testing. Results are available within 30 min. The Borrelia DotBlot G, although nearly comparable to the VIDAS in sensitivity and specificity, requires 2 h to complete, is more labor intensive, and is not designed for large-volume testing.

Replacement of two-tiered testing with a single test for serologic diagnosis of Lyme disease would be of great benefit for laboratories and health care providers. Less time would be required to complete testing, and proper antibiotic therapy would be provided more expeditiously.

The first-tier tests of conventional assays use lysate prepared from cell culture strains of B. burgdorferi. Although recombinant antigen assays, including chimeric antigen assays, have been developed and show promise, clinical studies are lacking to validate their use as stand-alone tests (3, 5, 6, 8). In fact, some of these assays have been demonstrated to be no more specific than conventional first-tier assays (7, 11), and others, though more specific when compared to conventional first-tier assays, lack sensitivity (5, 10). The wide variability of sensitivity and specificity of conventional first-tier tests has been demonstrated by numerous studies and likely relates to a lack of standardization methods for antigen preparation and validation (1, 4, 9). For example, unlike tests for human immunodeficiency virus, the commercially available serologic tests for antibody to B. burgdorferi are not standardized against a reference serum specimen panel (1). Until stand-alone tests with sufficient sensitivity and specificity are developed, a two-tiered approach for serologic diagnosis of Lyme disease is required. The results of the present study demonstrated that among the three first-tier EIA tests, the VIDAS test had the best performance and is the easiest to use.

 
* Corresponding author. Mailing address: Mayo Clinic, 200 First St. SW, Hilton 470, Rochester, MN 55905. Phone: (507) 284-2901. Fax: (507) 284-4272. E-mail: cockerill.franklin{at}mayo.edu.

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Journal of Clinical Microbiology, December 2002, p. 4782-4784, Vol. 40, No. 12
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.12.4782-4784.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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• Jobe, D. A., Lovrich, S. D., Schell, R. F., Callister, S. M. (2003). C-Terminal Region of Outer Surface Protein C Binds Borreliacidal Antibodies in Sera from Patients with Lyme Disease. CVI 10: 573-578 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jespersen, D. J. Articles by Cockerill III, F. R. Search for Related Content PubMed PubMed Citation Articles by Jespersen, D. J. Articles by Cockerill III, F. R.