KSHV DNA viremia correlates with low CD4+ cell count in Italian males at the time of diagnosis of HIV infection.

To evaluate the relevance and the virological and immunological markers of Kaposi sarcoma herpesvirus 8 (KSHV) viremia in Italian male patients at the time of diagnosis of infection with HIV-1, 481 men infected with HIV were recruited consecutively. The presence of KSHV DNA was evaluated in peripheral blood mononuclear cells (PBMCs) and in plasma and correlated with demographic and viro-immunological parameters. Seventy-four patients had KSHV DNA detected in PBMCs. By univariate analysis, the presence of KSHV DNA was associated significantly with unprotected homosexual relationships (P=0.003) and it was significantly higher in patients with CD4+ cell <350 (P=0.025). By multivariate analysis, homosexual relationships were associated independently with KSHV DNA in PBMCs (OR: 3.25; 95% CI: 1.1-9.7; P=0.035). Among the 74 patients with KSHV DNA detected in PBMCs, plasma samples from 60 were analyzed and 33 were positive for KSHV DNA. The CD4+ cell counts and percentages were significantly lower in patients with KSHV DNA in both PBMCs and plasma as compared to patients with only KSHV DNA in PBMCs (P=0.006 and P=0.019, respectively). Among the patients with KSHV DNA detected in PBMCs, all 13 patients with CD4+ cells count <200 had detectable levels of KSHV in their plasma. By multivariate analysis adjusted for the epidemiologic and virological parameters, low CD4+ cell count was the only independent variable associated with the presence of KSHV DNA in plasma (OR, 0.001; 95% CI: <0.001-0.001; P=0.03). In HIV-positive antiretroviral therapy-naïve males, KSHV active replication as detected by KSHV DNA in plasma was associated significantly with low CD4+ cell count.

The real-time polymerase chain reaction-guided modulation of immunosuppression enables the pre-emptive management of Epstein-Barr virus reactivation after allogeneic haematopoietic stem cell transplantation.

To assess a real-time polymerase chain reaction-based modulation of immunosuppression in patients with an increasing Epstein-Barr virus (EBV) viral load, we studied 79 paediatric allogeneic stem cell transplantations (allo-SCT) performed between January 1998 and December 2003. EBV reactivation was observed in 42 of 79 patients (53%) after a median time of 45 d from allo-SCT: 37 (88%) and five (12%) patients had received the graft from an unrelated and a related donor respectively (P = 0.001). Twenty-eight patients (67%) had a viral load > or =300 genomic copies x10(5) peripheral blood mononuclear cells (PBMC) and antithymocyte globulin was the only factor significantly associated with EBV reactivation (P = 0.001, RR 7.1). Among these 28 patients, immunosuppression was suspended and reduced in 17 and 11 patients respectively. Overall, post-transplant lymphoproliferative disease was diagnosed in one of 79 patients (1%). The pre-emptive modulation of immunosuppression in patients with EBV reactivation and a viral load > or =300 genomic copies x10(5) PBMC did not negatively influence transplant-related mortality, overall survival or event-free survival. In conclusion, EBV reactivation is frequent even in 'low risk' patients and the pre-emptive modulation of immunosuppression enables it to be managed safely, with no significant flare in graft-versus-host disease status.

Assessment of CMV load in solid organ transplant recipients by pp65 antigenemia and real-time quantitative DNA PCR assay: correlation with pp67 RNA detection.

After bone marrow (BM) or solid-organ (SO) transplantation viremic Cytomegalovirus (CMV) infection is observed frequently. Quantitative assay of CMV in blood helps the management of this clinical condition. In the present report, 83 samples from 39 solid organ recipients, three CMV assays were compared simultaneously for the first time: the Nuclisens CMV pp67 assay (nucleic acid sequence-based amplification, NASBA), an "in-house" quantitative real-time PCR assay (TaqMan) for CMV DNA, and pp65 antigenemia. The relation between CMV DNA and pp65 antigenemia, the quantitative assays, was evaluated on a larger group including 251 blood samples from 118 solid organ recipients. Real-time PCR provided the best results; > or =130 CMV DNA copies/2 x 10(5) peripheral blood leukocytes (PBLs) predicted > or =1 pp65 antigen positive (Ag+) cell/2 x 10(5) PBLs. By taking pp65 antigenemia as the "gold standard," the sensitivity of CMV DNA quantitation and of the pp67 RNA assay were 0.95 and 0.20, respectively, while the corresponding specificity values were 0.50 and 0.93. When real-time PCR was considered as the "gold standard," the sensitivity and specificity of the pp65 antigenemia were 0.65 and 0.91, respectively. Among the three tests examined, the sensitivity of the pp67 RNA assay was the lowest. On the other hand, the pp67 RNA assay was highly specific and effective in pinpointing high viremia patients. The present report, by providing predictive values for all three diagnostic profiles, DNA load, antigenemia, and pp67RNA, is a contribution for validation of real-time PCR as a new standard for quantitative assessment of CMV viremia in clinical settings.

The Sp1 transcription factor does not directly interact with the HIV-1 Tat protein.

The role of Sp1 in regulating the trans-activating activity of the human immunodeficiency virus type 1 (HIV-1) Tat protein has not yet been clearly defined. In fact, studies on the physical and functional interaction between Sp1 and Tat have yielded contradictory results. Here we investigated whether a physical interaction between Sp1 and Tat indeed occurs, exploiting both biochemical and genetic techniques that allow detection of direct protein-protein interactions. Studies performed with the yeast two-hybrid system indicate that Sp1 does not directly interact with the HIV-1 Tat protein. Control experiments demonstrated that both proteins are functionally expressed in the yeast cells. In vitro binding assays further confirmed that Sp1 does not physically bind Tat. These data suggest that in vivo Tat and Sp1 most likely take part of a multicomponent complex and thus encourage the search of the molecule(s) which mediate Tat-Sp1 interaction.