Use of epidemiologically well-defined subjects and existing immunofluorescence assays to calibrate a new enzyme immunoassay for human herpesvirus 8 antibodies

Abstract

Agreement between assays for the detection of human herpesvirus 8 (HHV- 8) antibodies has been limited. In part, this disagreement has been because assay calibration (i.e., differentiating positive from negative results) has not been done in a standardized fashion with reference to a wide spectrum of HHV-8-infected (true-positive) and HHV-8-uninfected (true-negative) persons. To describe the performance of an assay for HHV-8 antibodies more accurately, we used epidemiologically well-characterized subjects in conjunction with testing on two existing immunofluorescence assays for HHV-8 antibodies to define two groups: a group of 135 HHV-8-infected individuals (true positives), including Kaposi's sarcoma patients and those asymptomatically infected, and a group of 234 individuals with a high likelihood of being HHV- 8 uninfected (true negatives). A new enzyme immunoassay (EIA), using lysed HHV-8 virion as the antigen target, was then developed. With the above true positives and true negatives as references, the sensitivity and specificity of the EIA associated with different cutoff values were determined. At the cutoff that maximized both sensitivity and specificity, sensitivity was 94% and specificity was 93%. When the EIA was used to test a separate validation group, a distribution of seropositivity that matched that predicted for the agent of Kaposi's sarcoma was observed: 55% of homosexual men were seropositive, versus 6% seropositivity in a group of children, women, and heterosexual men. It is proposed that the EIA has utility for large-scale use in a number of settings and that the calibration method described can be used for other assays, both to more accurately describe the performance of these assays and to permit more-valid interassay comparison.