![]() Although the results are preliminary, microarrays for serologic detection of products of expressed open reading frames represent a promising new technology ( 3). ![]() While more specific than whole-cell EIAs, these new assays might not be as specific as Western blotting ( 5, 40). The predominant immune responses to C6 and VlsE1 are IgG mediated, even in early disease, while pepC10 generates an early and sometimes lasting IgM response ( 2, 5, 28). These surface antigens are expressed by Borrelia burgdorferi during the early phase of mammalian infection ( 39). The goal of the current study was to develop an objective alternative to Western blotting as a second-tier assay.ĭiagnostic serology has evolved and now utilizes recombinant and synthetic peptide antigens, such as C6, the 26-mer invariant portion of VlsE1 (variable major protein-like sequence 1) recombinant VlsE1 itself and pepC10, a 10-mer conserved portion of OspC ( 2). Western blotting is also labor-intensive and expensive. Although Western blotting is very sensitive for stage II and III disease, multiple limitations to blot accuracy have been identified: a low sensitivity for stage I disease, false-positive IgM immunoblots, and subjective interpretation of weakly positive bands ( 1, 5). Furthermore, only IgG blots were recommended for serologic diagnosis more than 30 days after disease onset. burgdorferi followed by supplementary IgG and IgM Western blotting of positive or indeterminate samples ( 9). ![]() ![]() Following the Second National Conference on the Serologic Diagnosis of Lyme Disease (27 to 29 October 1994 Dearborn, MI), a 2-tier serologic approach was recommended, comprised of an initial serum EIA or IFA for antibody to B. When first introduced for LD diagnosis, whole-cell enzyme immunoassays (EIAs) and indirect immunofluorescence assays (IFAs) for serum antibodies to Borrelia burgdorferi suffered from a lack of standardization, poor reproducibility, and high false-positive rates ( 11, 25). Overuse of serology has led to significant problems with false-positive results and misdiagnosis ( 38). Hinckley, Centers for Disease Control and Prevention, personal communication). Despite the predominance of stage I disease, more than 3.4 million tests for LD were ordered in 2008 in the United States (A. Diagnosis of stage I disease is based on clinical, not serological, criteria, while stages II and III typically require serologic confirmation ( 37). There are three disease stages: stage I is the early acute phase, characterized by a rash (erythema migrans ) that occurs in at least 70% of patients stage II represents early disseminated infection, including lymphocytic meningitis, cranial neuropathy, radiculopathy, and Lyme carditis and stage III represents late disseminated infection, such as Lyme arthritis, axonal peripheral neuropathy, and encephalomyelitis ( 39). Lyme disease (LD) is the most common vector-borne disease in the United States, with a reported incidence of nearly 35,000 new cases annually ( 10, 21). Prospective validation studies appear to be warranted. As a second-tier test, a multiplex assay for VlsE1-IgG and pepC10-IgM antibodies performed as well as or better than Western blotting for Lyme disease diagnosis. Compared to Western blotting, the multiplex assay was equally specific (95.6%) but 20.7% more sensitive for early-convalescent-phase disease (89.0% versus 68.3%, respectively 95% confidence interval for difference, 12.1% to 30.9%) and 12.5% more sensitive overall (75.0% versus 62.5%, respectively 95% CI for difference, 8.1% to 17.1%). A bioinformatic technique called partial receiver-operator characteristic (ROC) regression was used to combine individual antibody levels into a single diagnostic score with a single cutoff this technique enhances test performance when a high specificity is required (e.g., ≥95%). Our study population comprised 79 patients with early acute Lyme disease, 82 patients with early-convalescent-phase disease, 47 patients with stage II and III disease, 34 patients post-antibiotic treatment, and 794 controls. We developed an objective alternative second-tier immunoassay using a multiplex microsphere system that measures VlsE1-IgG and pepC10-IgM antibodies simultaneously in the same sample. Western blot accuracy is limited by subjective interpretation of weakly positive bands, false-positive IgM immunoblots, and low sensitivity for detection of early disease. The Centers for Disease Control and Prevention currently recommends a 2-tier serologic approach to Lyme disease laboratory diagnosis, comprised of an initial serum enzyme immunoassay (EIA) for antibody to Borrelia burgdorferi followed by supplementary IgG and IgM Western blotting of EIA-positive or -equivocal samples.
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