Detection of HHV-8 Associated with Kaposi’s Sarcoma

Human herpesvirus-8 (HHV-8) is a γ-herpesvirus which is related to the Epstein-Barr virus and is etiologically associated with Kaposi’s sarcoma (KS). The AIDS epidemic over the last 40 years has resulted in a high rate of KS due to immunosuppression. The seroprevalence for HHV-8 varies from 1-5% in the US to up to 80% in sub-Saharan Africa. A rapid, inexpensive diagnostic assay to detect HHV-8 would be very beneficial to screen those who may be potentially at risk for developing KS. Several immunological assays are available which require advanced instrumentation or technical staff to perform and analyze the results. At present, there is no lateral flow assay (LFA) available for HHV-8. The future development of an LFA for HHV-8 would be ideal in terms of patient confidentiality, analytical speed, and low cost.

One such available assay for HHV-8, described by Logan et al. (2016), is offered by MBio Diagnostics. Researchers measured the antibody reactivity for KS related HHV-8 (KSHV) in a collection of 164 clinical specimens from patients with biopsy-proven KS and sourced negative controls. The MBio system consists of disposable cartridges and a simple reader instrument, using planar waveguide imaging technology. The cartridge incorporates a microarray of recombinant antigens and antibody controls in a fluidic channel, providing multiple parallel fluorescence immunoassay results for a single sample. A protein microarray was printed to the planar waveguide and subsequently assembled into an injection-molded, disposable cartridge. The HIV/KSHV antibody detection cartridge comprised an array of three antigens: HIV-gp41 and KSHV antigens, LANA, and K8.1 The selection of these KSHV antigens was based on published observations for the primary reactivities to these proteins by patient sera. The array also included controls with anti-human IgG (sample control) and printed human IgG (detect reagent control). The samples were processed in cartridges on the bench top at room temperature. The median signal/cutoff (S/CO) for KSHV antibody reactivity was 3.17, which was 100-fold higher than the median S/CO of the controls with a median S/CO of 0.03. The median S/CO for the K8.1 antigen in the KS-positive samples was 7.26, which was > 200-fold higher than the median S/CO of the negative controls with a median S/CO of 0.-3. The MBio K8.1 assay showed an antibody reactivity for 162 of the 164 KS-positive samples and called 87 of 93 control samples as negative. The LANA assay reported three indeterminate results and reported antibody reactivity for 122 of the 161 KS-positive samples and called 89 of the 93 control samples as negative. The LANA has significantly lower sensitivity than K8.1. Researchers compared the MBio Diagnostic system to a commercially available, whole-virus ELISA kit by Advanced Biotechnologies, Inc., which detects anti-KSHV antibodies. KSHV antibody reactivity by ELISA was positive in 158 of 164 biopsy proven KS-positive samples. Comparing specific samples between MBio and ELISA, 3 of 8 MBio false negatives were also negative by ELISA. The ELISA has slightly better sensitivity in the KS-positive sample collection than the MBio assay.

The MBio KSHV assay correctly identified 155 of 164 plasma samples from patients with biopsy-proven KS and 85 of 93 KSHV antibody-negative samples for a sensitivity of 95.1% and specificity of 91.4%. The future development of a lateral flow assay for HHV-8 would be able to improve on these statistics, as well as to be more widely available to individuals and doctor’s offices on a confidential, cost conscious, basis.

By David Kilpatrick, PhD and Abbas Vafai, PhD

Logan, C., Todorof, K., Fiorillo, S. P., Campbell, T. B., Elder, J. H., Borok, M., Gudza, I., Gwanzura, L., Ndemera, B., Lochhead, M. J., Benson, C. A., & Schooley, R. T. (2016). Rapid multiplexed immunoassay for detection of antibodies to Kaposi’s sarcoma-associated herpesvirus. PloS One, 11(9).