Viral Load of Human Herpesvirus 8 in Peripheral Blood of Human Immunodeficiency Virus-Infected Patients with Kaposi's Sarcoma

Abstract

Kaposi's sarcoma (KS)-associated herpesvirus or Human herpesvirus 8 (HHV-8) (13), is a ␥-herpesvirus now widely established as a necessary cause of KS and also associated with body cavity-based lymphoma and multicentric Castleman's disease (5). These diseases were previously rare but are now brought to prominence by the AIDS pandemic. Detection of HHV-8 DNA in peripheral blood mononuclear cells (PBMCs) from human immunodeficiency virus type 1 (HIV-1)-infected persons is associated with an increased risk of subsequent development of KS (30,43) and with KS clinical stage (10, 11).Highly active antiretroviral therapy (HAART) is effective for inhibiting HIV replication, increasing CD4 cell counts, and delaying AIDS-associated opportunistic infections (26, 33). Case reports suggest that HAART may also be of benefit against AIDS-related KS as well as in eliminating detectable HHV-8 DNA from PBMCs of HIV carriers (15,24,28,35).Previous studies about the relationship between peripheral blood HHV-8 load and KS pathogenesis have been limited by the use of qualitative or semiquantitative estimates of HHV-8 load. Reproducible, sensitive, and specific quantitative techniques are needed to assess the HHV-8 DNA load and its correlation with different clinical conditions.We have therefore developed a highly sensitive and specific real-time PCR assay for the quantification of the HHV-8 genomes in peripheral blood. The present study wished to evaluate the consistency over time of HHV-8 viral DNA loads among KS patients receiving treatment for HIV-1 infection. We also sought to determine whether HHV-8 DNA viral load was correlated with HIV RNA viral load, CD4 cell counts, and/or serological reactivity to HHV-8. MATERIALS AND METHODSPatients. Fourteen HIV-infected patients with histologically confirmed KS (13 male and one female; range of ages, 28 to 56 years) were monitored at the Department of Oncology & AIDS, Centro di Riferimento Oncolgico of Aviano, Aviano, Italy, over the period 1997 to 2000. All patients but one had advanced clinical stages of KS disease, with visceral and/or lymph node involvement. According to the Krown staging system (21), 13 patients were at stage T1, 7 patients were at stage I1, and 8 patients were at stage S1.After informed consent was given by each patient, blood samples were collected at consecutive visits and HIV RNA viral load, CD4 T-cell count, HHV-8 serology for lytic and latent viral antigens and HHV-8 DNA viral load in peripheral blood were assessed. For each patient, one blood sample was collected before starting HAART, and then two or more samples were taken, depending on the number of control visits after the therapy had been initiated.Thirteen patients were receiving combination therapy with one protease inhibitor and two nucleoside reverse transcriptase inhibitors; one patient was receiving one nonnucleoside reverse transcriptase inhibitor and two nucleoside reverse transcriptase inhibitors.During the follow-up, three patients had also been treated with local radiotherapy; one patient had ...

Infection with Kaposi's sarcoma-associated herpesvirus (KSHV) and human immunodeficiency virus (HIV) in relation to the risk and clinical presentation of Kaposi's sarcoma in Uganda

“…These prospective data, together with the fact that the clinical burden of tumour lesions is independent of the titre of anti-KSHV antibodies (Table 1), argue against the possibility that high titres of antibodies against KSHV latent nuclear antigens result from the presence of Kaposi's sarcoma lesions. Presumably, the titre of antibodies against KSHV reflects the number of circulating KSHV-infected cells (i.e., the KSHV viral load), and that for a given titre, the burden of infected cells is higher among HIV-infected individuals than in those without HIV, and there is currently some evidence to support this (Tedeschi et al, 2001).…”

Maternal human polyomavirus infection and risk of neuroblastoma in the child

“…Each cell has a total DNA content of 6.7 pg, and by measuring of the DNA amount in the extracted material, the number of EBV genomes per microliter could be calculated. 20 The standard curve was created by the ABI PRISM 7700 Sequence Detection System software by plotting the Ct values against each known concentration of the BKV standard. The BKV standard virus stock (provided from Swedish Institute of Infectious Disease Control, Smittskyddsinstitutet, Solna, Sweden) originated from a urine sample from a BKV positive patient sampled 21 January 1987 and is thus designated as BKV strain 870121.…”

“…A threshold cycle value (Ct) is calculated for each sample by determining the point at which the fluorescence exceeds the threshold limit chosen for the specific plate. 19,20 BKV-specific primers and probe detecting the VP1-region of the BKV genome were designed using the Primer Express Software (PE Applied Biosystems, Cheshire, United Kingdom). The realtime PCR assay used the forward primer 5Ј-AGT GGA TGG GCA GCC TAT GTA-3Ј (nucleotide position 2511-2531) and the reverse primer 5Ј-TCA TAT CTG GGT CCC CTG GA-3Ј (nucleotide position 2605-2586) and the fluorogenic Taqman probe 5Ј-CTG TGC CAT CAA ACA CCC TAA CCT CTT CTA CCT-3Ј to amplify and detect a 94 bp amplicon within the VP1-region of the BKV genome.…”

Valutazione analitica e applicazione clinica di un metodo Real Time PCR per il dosaggio della carica virale di Epstein-Barr virus

“…L'interpretazione dei risultati è stata effettuata mediante risoluzione di 20 µl di ciascun prodotto di PCR dopo elettroforesi su gel di poliacrilammide e lettura dell'intensità relativa della fluorescenza dei frammenti colorati con etidio bromuro mediante densitometro (QuantityOne, BIORAD). Il sistema di quantificazione assoluta del DNA di EBV mediante Real Time PCR utilizza una sonda TaqMan marcata con due fluorocromi e l'enzima TaqGold polimerasi (23). Viene amplificato un frammento di 65bp del gene LMP-1 del genoma di EBV, mediante lo strumento ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems).…”