C6 Peptide-Based Multiplex Phosphorescence Analysis (PHOSPHAN) for Serologic Confirmation of Lyme Borreliosis

<div><p>Background</p><p>A single-tier immunoassay using the C6 peptide of VlsE (C6) from <i>Borrelia burgdorferi</i> sensu stricto (<i>Bb)</i> has been proposed as a potential alternative to conventional two-tier testing for the serologic diagnosis of Lyme disease in the United States and Europe.</p><p>Objective</p><p>To evaluate the performance of C6 peptide based multiplex Phosphorescence Analysis (PHOSPHAN) for the serologic confirmation of Lyme borreliosis (LB) in Russian patients.</p><p>Methods</p><p>Serum samples (<i>n</i> = 351) were collected from 146 patients with erythema migrans (EM); samples from 131 of these patients were taken several times prior to treatment and at different stages of recovery. The control group consisted of 197 healthy blood donors and 31 patients with other diseases, all from the same highly endemic region of Russia. All samples were analyzed by PHOSPHAN for IgM and IgG to <i>Bb</i> C6, recombinant OspC and VlsE proteins, and C6 peptides from <i>B</i>. <i>garinii</i> and <i>B</i>. <i>afzelii</i>.</p><p>Results</p><p>IgM and IgG to <i>Bb</i> C6 were identified in 43 and 95 out of 131 patients (32.8 and 72.5%, respectively); seroconversion of IgM antibodies was observed in about half of the patients (51.2%), and of IgG antibodies, in almost all of them (88.4%). Additional detection of OspC-IgM and VlsE-IgM or IgG to C6 from <i>B</i>. <i>garinii</i> or <i>B</i>. <i>afzelii</i> did not contribute significantly to the overall sensitivity of the multiplex immunoassay.</p><p>Conclusions</p><p>The multiplex phosphorescence immunoassay is a promising method for simultaneously revealing the spectrum of antibodies to several <i>Borrelia</i> antigens. Detection of IgM and IgG to <i>Bb</i> C6 in the sera of EM patients provides effective serologic confirmation of LB and, with high probability, indicates an active infection process.</p></div

Lyme Index values (LIV) for sera from LB patients with erythema migrans (N = 351) and healthy blood donors (N = 197).

<p>(A) PHOSPHAN variants M₁–M₃ and (B) PHOSPHAN variants G₁–G₃ and C6 ELISA (C6). Blood donors, *; line with arrowhead, threshold level (LIV = 1). Immunoassay codes: M₁, <i>Bb</i> C6 IgM; M₂, OspC IgM; M₃, VlsE IgM; G₁, <i>Bb</i> C6 IgG; G₂, <i>Bg</i> C6 IgG; G₃, <i>Ba</i> C6 IgG; C6, Immunetics C6 Lyme ELISA kit. (<i>Bb</i>) <i>B</i>.<i>burgdorferi</i>, (<i>Bg</i>) <i>B</i>.<i>garinii</i>, (<i>Ba</i>) <i>B</i>.<i>afzelii</i>. Legend: Transparent square, Mean; Spotted rectangle, ± Standard Error; Dotted rectangle, Non-Outlier Range; Transparent circle, Outliers; Black square, Extremes.</p

Sensitivity of PHOSPHAN tests for serum IgG antibody responses to <i>B</i>. <i>burgdorferi</i> C6, <i>B</i>. <i>garinii</i> C6, and <i>B</i>. <i>afzelii</i> C6 in samples from EM patients.

<p>(A) Total sensitivity of PHOSPHAN variants G₁, G₂, G₃, and G_1–3 at the baseline prior to treatment (N = 146). (B) Sensitivity of PHOSPHAN variants G₁–G₃ in tests of samples taken at the baseline (<i>n</i> = 146) and on days 8–14 (<i>n</i> = 75), 15–30 (n = 48), and 31–66 (<i>n</i> = 82) after disease onset. Values that are statistically significant (Fisher's exact test, <i>p</i> < 0.05) are denoted by brackets or asterisks. Immunoassay codes: G₁, <i>Bb</i> C6 IgG; G₂, <i>Bg</i> C6 IgG; G₃, <i>Ba</i> C6 IgG; G_1–3, any of the three antigens <i>Bb</i> C6, <i>Bg</i> C6, or <i>Ba</i> C6. (<i>Bb</i>) <i>B</i>.<i>burgdorferi</i>, (<i>Bg</i>) <i>B</i>.<i>garinii</i>, (<i>Ba</i>) <i>B</i>.<i>afzelii</i>. Legend: (B) Transparent square, G1; Black diamond, G2; Black triangle, G3.</p

Sensitivity of PHOSPHAN and C6 ELISA tests for serum IgM and IgG antibody responses to <i>B</i>. <i>burgdorferi</i> C6 in samples from EM patients.

<p>(A) Sensitivity of PHOSPHAN variants M₁, G₁, M₁G₁ and C6 ELISA (C6) at the baseline prior to treatment (N = 146). (B) Sensitivity of PHOSPHAN variants M₁ and G₁, and (C) PHOSPHAN variant M₁G₁ and C6 ELISA in tests of serum samples taken at the baseline (n = 146) and on days 8–14 (<i>n</i> = 75), 15–30 (n = 48), and 31–66 (<i>n</i> = 82) after disease onset. Values that are statistically significant (Fisher's exact test, <i>p</i> < 0.05) are denoted by brackets or asterisks. Immunoassay codes: M₁, <i>Bb</i> C6 IgM; G₁, <i>Bb</i> C6 IgG; M₁G₁, <i>Bb</i> C6 IgM+IgG; C6, Immunetics C6 Lyme ELISA kit. (<i>Bb</i>) <i>B</i>.<i>burgdorferi</i>. Legend: (B) Transparent square, M1; Black diamond, G1. (C) Black square, M1G1; Transparent circle, C6.</p