Improved serodiagnostic testing for Lyme disease: results of a multicenter serologic evaluation.

The diverse clinical manifestations of Lyme disease (1-3) have led to frequent confusion in clinical diagnosis, a confusion compounded by problems in the accuracy and precision of diagnostic serologic tests (4-11) and the difficulty of isolating the causative organism (12-14), Borrelia burgdorferi. In 1990, more than 20 commercially prepared serologic test kits for Lyme disease were being sold in the United States, but no nationally standardized reference test was available. A collaborative evaluation of a selected sample of the commercial test kits by the Centers for Disease Control and Prevention (CDC) and the Association of State and Territorial Public Health Laboratory Directors (ASTPHLD) demonstrated poor concordance of results among these test kits and among a selected group of state health department laboratories (11). Because of the lack of a rigorously defined reference serum panel, conclusions could not be drawn about the sensitivity and specificity of the test kits evaluated. An unexpected finding in this study was the low concordance in test results between CDC and two consulting academic reference center laboratories. A number of other studies also have demonstrated low concordance of Lyme disease serologic test results obtained by a variety of laboratories (4-10). As a result of those findings,the study described here was designed to fulfill the following objectives: 1) to assemble a serum panel from patients who had clinically well-defined Lyme disease (preferably confirmed by isolation of B. burgdorferi); healthy controls, and persons residing in non–endemic-disease areas whose potentially cross-reactive specimens had yielded equivocal ELISA results in earlier CDC tests; 2) to test this panel in a blinded fashion by several recognized Lyme disease reference and research laboratories; and 3) to compare the accuracy and precision of tests as a prelude to developing national recommendations for standardized serologic testing for antibodies to B. burgdorferi. Tests were performed by five academic centers active in Lyme disease research (the Marshfield Clinic, Marshfield, Wisconsin; University of Medicine and Dentistry of New Jersey–Robert Wood Johnson Medical School, New Brunswick, New Jersey; State University of New York at Stony Brook, Stony Brook, New York; Tufts/New England Medical Center, Boston, Massachusetts; and the University of Connecticut Health Center, Farmington, Connecticut) and CDC’s Division of Vector-Borne Infectious Diseases,National Center for Infectious Diseases, based in Ft. Collins, Colorado. Serum samples from Lyme disease case-patients were obtained from the participating academic investigators (n = 72) and from the CDC Lyme disease reference serum collection (n = 37). All case-patient serum samples (total = 109) were from patients who met the CDC clinical case definition for surveillance of Lyme disease (15). The clinical manifestations in these patients ranged from acute erythema migrans (EM) to late neurologic disease accompanied by Lyme arthritis. B. burgdorferi had been cultured by the method of Berger et al. from 14 of 34 (41%) acute-phase specimens provided by CDC (14). Duplicate specimens (n = 85) were randomly selected from the 109 case-patient samples for precision analysis, making a total of 194 case-patient samples in the panel. Control serum samples were provided by CDC from unpaid healthy blood donors (n = 113) who resided in areas where Lyme disease is not endemic (Cincinnati, Ohio, and Atlanta, Georgia; travel histories were not available from these donors, however. Duplicate specimens (n = 87) also were randomly selected, resulting in 200 noncase samples in the serum panel. Additional control samples were obtained from persons who resided in areas where Lyme disease was not endemic but whose physicians submitted their serum for Lyme disease testing to CDC through their state health department (n = 113). These specimens from patients with suspected cases had borderline (equivocal) seroreactivity in the whole cell sonicate (WCS) enzyme-linked immunoassay (ELISA) used by CDC before 1992 and are referred to hereafter as “WCS-suspects” (16). The addition of duplicate specimens (n = 87) brought this group to 200 equivocally seroreactive samples. Serum was separated and frozen by the original collectors and shipped frozen to CDC’s facilities in Ft. Collins, Colorado. The specimens were divided into aliquots and coded; code labels were applied Dispatches