Effect of Borrelia burgdorferi Genotype on the Sensitivity of C6 and 2-Tier Testing in North American Patients with Culture-Confirmed Lyme Disease

MAJOR ARTICLE Effect of Borrelia burgdorferi Genotype on the Sensitivity of C6 and 2-Tier Testing in North American Patients with Culture-Confirmed Lyme Disease Gary P. Wormser,1 Dionysios Liveris,2 Klára Hanincová,2 Dustin Brisson,4 Sara Ludin,1 Vincent J. Stracuzzi,1 Monica E. Embers,5 Mario T. Philipp,5 Andrew Levin,6 Maria Aguero-Rosenfeld,3 and Ira Schwartz2 1 Background. A potential concern with any serologic test to detect antibodies to Borrelia burgdorferi is whether the epitopes incorporated in the test provide sufficient cross-reactivity to detect infection with all of the pathogenic strains of the species. This is a particular concern for the C6 test, which is based on reactivity to a single peptide. Methods. C6 testing and 2-tier testing were performed on acute-phase serum samples obtained from 1158 patients with erythema migrans for whom the genotype of the borrelial isolate was defined on the basis of an analysis of the 16S-23S ribosomal DNA spacer region and/or on the genetic variation of the outer surface protein C gene (ospC). The sonicated whole cell–based enzyme-linked immunosorbent assay, the immunoblots used in the 2-tier testing, and the C6 assay all used antigens from B. burgdorferi sensu stricto strain B31. Results. The sensitivity of C6 testing (69.5%) was greater than that of 2-tier testing (38.9%) (P ! .001); the difference in sensitivity, however, was statistically significant only for patients infected with 2 of the 3 ribosomal spacer type–defined genotypes. The lower sensitivity of 2-tier testing was attributable to the low sensitivity of the immunoblot tests, rather than the first-tier enzyme-linked immunosorbent assay. There was also a trend for the sensitivity of 2-tier testing to vary according to the ospC genotype for the 14 genotypes represented in the study (P p .07); this relationship was not observed with C6 testing. Conclusions. Lack of sensitivity of the C6 test because of strain diversity seems less likely to be a limitation of this serologic test, compared with 2-tier testing in North American patients with early Lyme disease. Detection of antibody to C6, a 26–amino acid peptide that reproduces the sequence of the sixth invariable region (IR6) within the central domain of the VlsE protein of Borrelia burgdorferi sensu lato, is currently used for the serologic diagnosis of Lyme disease [1–4]. A potential concern with a test based on a single peptide is the possibility of sequence variation and a consequent lack of antigenic cross-reactivity among pathogenic Received 3 April 2008; accepted 20 May 2008; electronically published 22 August 2008. Reprints and correspondence: Dr. Gary P. Wormser, Rm. 245, Munger Pavilion, New York Medical College, Valhalla, NY 10595 (). Clinical Infectious Diseases 2008; 47:910–4  2008 by the Infectious Diseases Society of America. All rights reserved. 1058-4838/2008/4707-0008$15.00 DOI: 10.1086/591529 910 • CID 2008:47 (1 October) • Wormser et al. strains of B. burgdorferi sensu lato, leading to reduced diagnostic sensitivity in some infected patients. B. burgdorferi sensu stricto (hereafter referred to as B. burgdorferi) isolates can be categorized into 3 distinct ribosomal spacer restriction fragment length polymorphism genotypes (RSTs) on the basis of analysis of the 16S-23S ribosomal DNA (rDNA) spacer region [5, 6]. North American isolates of B. burgdorferi can also be differentiated into at least 16 major genetic groups on the basis of variability in the genetic sequence encoding outer surface protein C (OspC) [7, 8]. Studies employing these methods have demonstrated that pathogenicity is dependent at least in part on the genotype of B. burgdorferi causing the infection; for example, evidence indicates that RST1 and RST2 are more likely to be associated with hematogenous dissemination in humans than is RST3 [6]. Division of Infectious Diseases, Department of Medicine, and Departments of 2Microbiology and Immunology and 3Pathology, New York Medical College, Valhalla; 4Department of Biology, University of Pennsylvania, Philadelphia; 5Division of Bacteriology and Parasitology, Tulane National Primate Research Center, Tulane University Health Sciences Center, New Orleans, Louisiana; and 6Immunetics, Boston, Massachussetts Although serologic testing of patients with erythema migrans is not routinely recommended for the management of these patients, serum samples from such patients are often used to validate or compare serologic assays, because this is the most common manifestation of Lyme disease [9], and it is the only manifestation for which the diagnosis can frequently be confirmed by the microbiologic gold standard of recovering B. burgdorferi on culture [10]. In a previous study [11], we demonstrated the comparable sensitivity of a C6 assay in 79 patients from North America with erythema migrans, irrespective of the RST genotype that caused the infection. The current study expands on the previous study by increasing the number of patient serum samples evaluated and by also considering the ospC genotype of the infecting strain of B. burgdorferi. In addition, the effect of the genotype of B. burgdorferi on the sensitivity of 2-tier testing was evaluated. Serologic assays. The C6 Lyme ELISA kit (Immunetics), a test kit approved by the US Food and Drug Administration, was used according to the manufacturer’s instructions. In place of the original cutoff formula that was based on a serum sample positive for Lyme disease, a simplified cutoff formula based on a negative calibrator serum sample was employed. The assay cutoff value was determined by adding 0.3 to the absorbance value of the calibrator serum sample. The Lyme index value for each patient sample was calculated by dividing the absorbance of the sample at 450–650 nm by the cutoff value. This modification yielded statistically equivalent sensitivity and specificity to what had been demonstrated with the original cutoff formula; the original cutoff formula was chosen to yield a specificity of 99.6% (95% CI, 97.9–100%) in a group of blood donors from endemic and nonendemic areas and a sensitivity of 85.8% in a combined group of patients with early- and latestage Lyme disease (A.L., unpublished data). The C6 peptide used as the antigen in the C6 Lyme ELISA kit is derived from the B. burgdorferi B31 strain sequence, which differs from the originally described IP90 Borrelia garinii sequence by 4 amino acids. The kit is formatted as an indirect ELISA in which both IgG and IgM antibodies to the C6 peptide are detected by an enzyme conjugate. Two-tier serologic testing was performed using an IgG-IgM ELISA kit (Wampole Laboratories), followed by Lyme IgG and IgM immunoblot kits (MarDx/Trinity Biotech). The 2-tier ELISA and immunoblot kits were approved by the US Food and Drug Administration for in vitro diagnostic use, and testing was performed according to the manufacturers’ instructions. Both the ELISA and immunoblot kits used a sonic (...truncated)