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

Agreement between assays for the detection of human herpesvirus 8 (HHV-8) a ntibodies has been limited. In part, this disagreement has been because ass ay calibration (i.e., differentiating positive front negative results) has not been done in a standardized fashion with reference to a wide spectrum o f HHV-8-infected (true-positive) and HHV-8-uninfected (true-negative) perso ns. To describe the performance of an assay for HHV-8 antibodies more accur ately, we used epidemiologically well-characterized subjects in conjunction with testing on two existing immunofluorescence assays for HHV-8 antibodie s to define two groups: a group of 135 HHV-8-infected individuals (true pos itives), including Kaposi's sarcoma patients and those asymptomatically inf ected, and a group of 234 individuals with a high likelihood of being HHV-8 uninfected (true negatives). A new enzyme immunoassay (EW), using lysed HH V-8 virion as the antigen target, was then developed. With the above true p ositives 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 validati on group, a distribution of seropositivity that matched that predicted for the agent of Kaposi's sarcoma was observed: 55% of homosexual men were sero positive, versus 6% seropositivity in a group of children, women, and heter osexual 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 thes e assays and to permit more-valid interassay comparison.