DETECTION OF HUMAN HERPES VIRUS 8 (HHV-8) IN BODY FLUIDS USING REAL TIME QUANTITATIVE POLYMERASE CHAIN REACTION ASSAYS

Abstract

Since the discovery of human herpes virus 8 ((HHV-8) in 1994, several serological and molecular screening assays have been developed to detect HHV-8 in tissues and body fluids. In order to quantitate and improve detection of low viral HHV-8 copies in clinical samples, the recently developed technique of quantitative PCR, utilizing TaqMan chemistry, was used to develop two assays that target the HHV-8 K6, viral macrophage inflammatory protein alpha (v-mip I), and open reading frame 22 (ORF 22), glycoprotein H, regions of the virus. These regions represent both a non-structural and a structural component, respectively, of the HHV-8 virus, and one assay can be used to confirm the other in cases of extreme low viral copy number. The TaqMan assays were compared with a published nested PCR method that detects HHV-8 open reading frame 26 (ORF 26). The results prove that the TaqMan and nested PCR assays have comparable sensitivity and specificity. Peripheral blood mononuclear cells (PBMC) samples from Kaposi's sarcoma (KS) endemic and epidemic populations were screened to investigate prevalence and viral load levels in these high- risk HHV-8 populations. The results suggest that the viral load in the KS endemic population is significantly higher than in the epidemic population. However, the prevalence of HHV-8 detected in these populations is almost identical. Quantification of HHV-8 viral load, using the new assays, in PBMC, saliva, and cervical secretions from women who differ in sexual practices, did not show significant differences between the two cohorts. Enzyme linked immunosorbant assays and immunofluorescence assay results with these same individuals do suggest a correlation between increased sexual contacts and HHV-8 positivity in the Spanish prostitute cohort. Evidence of HHV-8 transmission by heterosexual sex has been conflicting in non-KS endemic regions. Analysis of risk factors among the women working as prostitutes have identified human papillomavirus infection and age at first oral sex experience as independent risk factors of HHV-8 positivity. These correlations imply that sexual transmission of HHV-8 occurs in the Spanish prostitute cohort. Molecular detection of HHV-8 in PBMC samples is related to seropositivity, however the results indicate that HHV-8 sequence is also found in cervical and saliva samples from seronegative women. Both real-time PCR assays detect HHV-8 more frequently in samples from high-risk populations, which include Human Immunodeficiency virus-infected (HIV) patients and individuals from the KS endemic region of Uganda.