Comparative Study of Kaposi's Sarcoma-Associated Herpesvirus Serological Assays Using Clinically and Serologically Defined Reference Standards and Latent Class Analysis

Abstract

Accurate determination of infection with Kaposi's sarcoma-associated herpesvirus (KSHV) has been hindered by the lack of a "gold standard" for comparison of serological assays used to estimate KSHV prevalence in serosurveys conducted in different settings. We have evaluated the performance of five in-house (developed at University College London [UCL], United Kingdom, and at the virology laboratory of the Instituto de Medicine Tropical [IMT] in Sao Paulo, Brazil) and two commercial (ABI and DIAVIR) serological assays to detect antibodies to latency-associated nuclear antigen (LANA) and to lytic KSHV antigens. We used a variety of serum samples assembled to represent populations likely to be at high, intermediate, and low risk of KSHV infection in Brazil. Composite reference standard panels were prepared based on clinical and serological parameters, against which assay performances were assessed using conventional Bayesian statistics and latent class analysis (LCA). Against the clinical reference standard, in-house immunofluorescence assays to detect anti-LANA antibodies (IFA-LANA) produced at UCL and IMT had similar performances, with sensitivities of 61% (95% confidence interval [CI], 48% to 74%) and 72% (95% CI, 58% to 83%) and specificities of 99% (95% CI, 94% to 100%) and 100% (95% CI, 96% to 100%), respectively, and only the IMT IFA-LANA was included in LCA, together with the IMT IFA-lytic and four enzyme-linked immunosorbent assays (ELISAs). The LCA indicated that the IMT whole-virus ELISA performed best (sensitivity, 87% [95% CI, 81% to 91%]; and specificity, 100% [95% CI, 98% to 100%]), confirming the results obtained with the conventional statistical approach. Commercially available ELISA-based tests yielded the lowest specificities using a spectrum of serum samples. The evaluation of KSHV serological assays is warranted before planning serosurveys in various settings.

Prospective study of human herpesvirus 8 oral shedding, viremia, and serological status among human immunodeficiency virus seropositive and seronegative individuals in Sao Paulo, Brazil

“…The HHV-8 whole-virus ELISA assay had the highest sensitivity, confirming previous reports [9,11,14], a moderate specificity, and a high NPV. The combination with IFA-LANA assay increased specificity without losing much on sensitivity and had the highest PPV to detect shedders.…”

“…The lack of a gold standard for the serological diagnosis of HHV-8 remains an obstacle for comparing the sensitivity and specificity of each assay, making it difficult to establish what is the most reliable assay to discriminate true HHV-8 infected individuals from those who are not infected [14]. By testing six sequential serum samples blindly from each patient, the present study was able to show excellent intra-assay concordance for all three techniques.…”

“…Six serum samples from each patient were tested without knowledge of other results in a random patient and samples order for HHV-8 serologies at the Laboratory of Virology, Institute of Tropical Medicine, University of Sao Paulo, using in-house immunofluorescence assays (IFA) to identify antibodies to latent and lytic HHV-8 viral proteins, and a whole-virus HHV-8 enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G detection, as described previously [14]. …”

“…The lack of a gold standard for HHV-8 serological status further compounds the limited ability to determine ‘true infection’ status [14]. …”

Seroprevalence of Kaposi Sarcoma–associated Herpesvirus and Other Serologic Markers in the Brazilian Amazon

“…In the absence of a defi nitive test to determine KSHV infection, all serum specimens were tested by using a previously validated in-house whole-virus KSHV ELISA (12) and 2 immunofl uorescence assays (IFAs) that detected antibodies against lytic (IFA-lytic) and latent-associated nuclear antigens (IFA-LANA) (12). KSHV infection was defi ned as positivity by any of these serologic assays.…”