Calibrated Real-Time PCR Assay for Quantitation of Human Herpesvirus 8 DNA in Biological Fluids

Abstract

Accurate laboratory tests for the diagnosis of active human herpesvirus 8 (HHV-8) infection are becoming essential to study the pathogenesis of HHV-8-associated tumors and for the clinical management of HHV-8-infected individuals. We have developed a highly sensitive, calibrated quantitative real-time PCR assay for the measurement of cell-free HHV-8 DNA in body fluids, based on the addition of a synthetic DNA calibrator prior to DNA extraction. The calibrator controls each sample for the presence of PCR inhibitors, determines a cutoff value of sensitivity for negative samples, and normalizes positive samples for the efficiency of DNA recovery. The assay shows a wide dynamic range of detection (between 1 and 10 6 viral genome equivalents/reaction) and a high degree of accuracy even in the presence of high amounts (up to 1 g) of human genomic DNA. Moreover, the assay has a very high sensitivity (lower detection limit, 10 genome equivalents/ml) and a high degree of reproducibility and repeatability with a coefficient of variation (CV) of <15 and 23%, respectively. Furthermore, the use of the calibrator improves the accuracy of quantitation and decreases the intersample variability (CV, 9 and 6%, respectively). The sensitivity and specificity of the assay were tested with a series of clinical specimens obtained from patients affected by various HHV-8-related diseases, as well as from a wide number of controls. In conclusion, our calibrated real-time PCR assay provides a reliable high-throughput method for quantitation of HHV-8 DNA in clinical and laboratory specimens.Human herpesvirus 8 (HHV-8) is a recently characterized gamma herpesvirus associated with the development of Kaposi's sarcoma (KS), body-cavity-based-lymphoma (BCL), and some forms of multicentric Castleman's disease (MCD) (6,14,40). This virus is distributed worldwide with five distinct subtypes (A to E); yet, unlike other human herpesviruses, HHV-8 is not ubiquitous. Sero-epidemiological studies demonstrated a low prevalence of anti-HHV-8 antibodies in the general population in the United States and Asia (1 to 2%), an intermediate seroprevalence in Italy and other Mediterranean countries (5 to 35%), and the highest seroprevalence in Southern and Central Africa (30 to 60%) and among Brazilian Amerindians (41 to 65%) (1,4,8,19,31,35,38,39,46). HHV-8 seroconversion is associated, in some instances, with KS development (9). In addition, HHV-8 seropositivity can predict progression to KS in human immunodeficiency virus (HIV)-seropositive homosexual men (13,26,33).A variety of serological assays for diagnosis of HHV-8 infection have been described, but most of them fall short in sensitivity, specificity, and interassay concordance. Indeed, a combination of several assays should be utilized in order to obtain the optimal sensitivity and specificity (7,35,41). Moreover, the assessment of titers of antibody in serum may not always provide useful clinical information, especially for the therapeutic management of patients, since it does not allow distinguishing ...

Interactions between Human Immunodeficiency Virus Type 1 and Vaccinia Virus in Human Lymphoid Tissue Ex Vivo

“…Data were analyzed using sequence detection software (Applied Biosystems). We performed VACV DNA quantification using a commercially available reference standard (Advanced Biotechnologies, Columbia, MD) and normalized the results using a synthetic DNA calibrator molecule (10 6 copies per sample) that was added to the samples before the extraction step; this procedure allowed us to control for intersample extraction efficiency and to monitor PCR artifacts (4). Primers and the calibrator probe were not cross-reactive with the VACV sequences.…”

Productive infection of HUVEC by HHV-8 is associated with changes compatible with angiogenic transformation

“…Data were normalized based on the recovery rate of the calibrator DNA as described (Broccolo et al, 2002).…”

Evidence for both Lytic Replication and Tightly Regulated Human Herpesvirus 8 Latency in Circulating Mononuclear Cells, with Virus Loads Frequently below Common Thresholds of Detection

“…In EBVinfected individuals with PTLD, these virus genome loads would be considered low (29). Even though EBV-infected lymphoblasts proliferate in lymph nodes and circulating memory B cells are only latently infected, PTLD patients have EBV genome frequencies of 5,000 to 50,000 copies per 10 6 PBMCs at the time of diagnosis (19,28). Furthermore, the frequency of infected cells in PCR-positive KS subjects (0.84 to 29 per million PBMCs) is low enough to overlap with the higher end of the range of frequencies of EBV latently infected cells in healthy carriers (0.1 to 24 per million PBMCs) (37), again emphasizing the biological differences between the two human gammaherpesviruses.…”

“…The lower detection rate in plasma than in PBMCs is probably due at least in part to a sample volume issue: while 200 l of plasma was tested (representing approximately 0.4 ml of blood), the PBMCs tested in the 12 replicate reactions represented a total blood volume of 0.4 to 2.1 ml. In some studies reporting high detection rates in plasma or serum, virus particles were concentrated by centrifugation prior to DNA extraction (6,36).…”

“…Thus far, studies by others have focused on the number of HHV-8 genomes per volume of blood or per quantity of PBMCs (4,6,8,23,33). The frequency of infected blood cells or the number of HHV-8 genomes per infected cell, fundamental metrics in answering some of the outstanding questions outlined above, has not been measured.…”