Real-Time PCR for Quantification of Human Herpesvirus 6 DNA from Lymph Nodes and Saliva

A real-time quantitative PCR assay has been developed to measure human herpesvirus 6 (HHV-6) DNA in biological specimens. The assay sensitivity was 10 copies of DNA per well, with a linear dynamic range of 10 to 10(7) copies of HHV-6 DNA. Intra- and interassay variations were, respectively, 0.88 and 0.8% for samples containing 10(2) DNA copies, 0.99 and 0.96% for samples containing 10(4) copies, and 0.76 and 0.9% for samples containing 10(6) copies. Among 34 saliva samples from healthy subjects, 26 were found to contain HHV-6 DNA (76.5%; median, 23,870 copies/ml), and following a single freeze-thaw cycle, 25 of the same samples were found to be positive for HHV-6 DNA, although at a statistically significantly lower concentration (median, 3,497 copies/ml). The assay enabled detection of HHV-6 DNA in lymph node biopsies from patients with Hodgkin's disease (HD) (13 of 37 patients [35.1%]), B-cell neoplasms (8 of 36 patients [22.2%]), and T- or NK-cell neoplasms (3 of 13 patients [23.1%]), with concentrations ranging from 100 to 864,640 HHV-6 copies per μg of DNA (HHV-6B being found in every case except two). All HD patients infected with HHV-6 presented clinically with the nodular sclerosis subtype of HD. The real-time quantitative PCR assay developed here was simple to perform and was sensitive over a wide range of HHV-6 concentrations. It therefore appears to be of potential value in clinical investigation or diagnosis of HHV-6 infection

Specific antibody production in herpes keratitis: intraocular inflammation and corneal neovascularisation as predicting factors

International audiencePURPOSE: The purpose of the study is to investigate whether analysis of specific antibody synthesis can aid the diagnosis of herpes keratitis. METHODS: Aqueous humor was collected from 39 patients with presumed recurrent herpes keratitis, including 23 consulting for keratitis and 16 patients scheduled for penetrating keratoplasty. Local antibody production was ascertained by analysis of paired aqueous humor/serum samples, using a modified micro-ELISA technique. RESULTS: Local production of antibodies was found in 32 patients (82%): anti-herpes simplex virus (HSV) antibodies in 26 (67%) and anti-varicella zoster virus (VZV) antibodies in 11 (28%). Twenty of 23 patients with active keratitis (87%), and 12 of 16 undergoing keratoplasty (75%), tested positive. Five patients had local production of both anti-HSV and anti-VZV antibodies, whereas seven patients tested negative. Local antibody production was significantly associated with intraocular inflammation (P<0.05), corneal neovascularisation (P<0.05), and positive response to anti-viral treatment (P<0.05). No complications were encountered in sampling aqueous humor. CONCLUSIONS: Assessment of local anti-HSV and -VZV antibody production is a safe and reliable diagnostic procedure for recurrent herpes keratitis. It might be particularly helpful in patients presenting with intraocular inflammation and neovascularisation since it discriminates between herpes and non-herpes pathologies and may therefore be useful for preventive and therapeutic strategies