Evaluation of an automated extraction system in combination with Affigene CMV Trender for CMV DNA quantitative determination: comparison with nested PCR and pp65 antigen test.

CMV viral load quantitation is a powerful tool to assist clinicians in making accurate diagnoses, managing post-transplant CMV disease and monitoring antiviral therapy. The aim of this study was to evaluate the performance of Affigene CMV Trender for CMV viral load determination used in combination with a non-dedicated nucleic acid extraction system (BioRobot MDx) for high-throughput routine. Linearity, reproducibility and sensitivity were examined. Clinical samples were used to compare results obtained with the Affigene CMV Trender, with an "in house" nested PCR used for routine diagnosis and with pp65 antigenemia. The results indicated that the test is linear in the range of 1.81-5.18 Logcopies/ml and that sensitivity is 77 copies/ml. The concordance of the Affigene CMV Trender with nested PCR was high, (k=0.91, IC 95%=0.82-1.00), whereas a substantial concordance with pp65 antigenemia was observed (k=0.64, IC 95%=0.54-0.73). In conclusion, combined use of a non-dedicated automated nucleic acid extraction method with the Affigene CMV Trender results in an accurate high throughput system, suitable for routine laboratory monitoring of CMV infection.

Comparison of Versant HBV DNA 3.0 and COBAS AmpliPrep-COBAS TaqMan assays for hepatitis B DNA quantitation: Possible clinical implications.

We compared two commercial assays for HBV DNA quantitation, Versant HBV 3.0, System 340 (bDNA; Bayer Diagnostics) and COBAS AmpliPrep-COBAS TaqMan HBV Test (TaqMan; Roche Diagnostics). Analytical sensitivity, calculated on WHO International Standard, predicted 95% detection rate at 11.4 and 520.2IU/ml for TaqMan and bDNA, respectively. Specificity, established on 50 blood donor samples, was 100% and 84% for TaqMan and bDNA, respectively. When using clinical samples, HBV DNA was detected by TaqMan in 21/55 samples negative to bDNA. Mean values of HBV DNA obtained with bDNA were higher than those obtained with TaqMan (4.09log(10)+/-1.90 versus 3.39log(10)+/-2.41, p<0.001), and 24.4% of samples showed differences in viral load values >0.5log(10), without association with HBV genotype. There was a good correlation for HBV DNA concentrations measured by the two assays (r=0.94; p<0.001) within the overlapping range, and the distribution of results with respect to relevant clinical threshold recently confirmed (20,000 and 2000IU/ml) was similar. Approximately 50% of samples with low HBV DNA, appreciated by TaqMan but not by bDNA, were successfully sequenced in pol region, where drug resistance mutations are located.

Heterogeneity of HVR-1 quasispecies is predictive of early but not sustained virological response in genotype 1b-infected patients undergoing combined treatment with PEG- or STD-IFN plus RBV.

ISDR mutation pattern and HVR-1 quasispecies were analyzed in HCV genotype 1b-infected patients treated with either PEG- or STD-IFN plus ribavirin, in order to find virological correlates of therapy outcome. ISDR region analysis, performed at baseline (T0) and at 4 weeks of therapy (T1), indicated that ISDR mutation pattern was not predictive of response to treatment. Moreover, no selection of putative resistant strains in the first month of therapy was observed. Viral load was not correlated with any parameter of HVR-1 heterogeneity. Among the HVR-1 heterogeneity parameters considered, complexity was inversely correlated to viral load decline at T1. In univariate analysis, complexity, proportion of non synonymous substitutions (NS) and NS/S ratio were lower in patients showing virological response at 6 months of treatment. Complexity was the only parameter independently associated with both decline of viral load at T1 and virological response after 6 months, even after adjustment for confounding variables. At the end of treatment or later, these correlations were lost. Evolution pattern of the HVR-1 quasispecies indicated a strong selective pressure in sustained responders, with complete substitution of pre-existing quasispecies, while minor changes occured in non responders. In relapsers both patterns were present at a similar rate. In conclusion, this study shows that HVR-1 heterogeneity may be involved in the early response to combined IFN-RBV therapy. The loss of correlation between viral heterogeneity and therapy outcome at 6 months of therapy, or later, suggests that other factors may play a role in maintaining sustained response to treatment.

Significant reduction in HIV-1 plasma viral load but not in proviral infected cells during sub-optimal antiretroviral therapy.

Attempts to eradicate HIV infection through highly active antiretroviral therapy (HAART) in the very early stages of the infection have failed due to the resumption of viral replication from unknown reservoirs. It has been postulated that antiretroviral therapy capable of suppressing viral replication, as shown by reduction of HIV-RNA copies in plasma and lymph nodes, should have less effect on the number of HIV-DNA carrying cells in the same districts. To test this hypothesis, plasma viremia and the proportion of provirally infected cells in peripheral blood and in lymph nodes were measured in patients at 3 and 6 months of treatment with zidovudine plus lamivudine. All patients showed a significant decrease in plasma viremia at 3 months that was maintained at 6 months (mean values of 1.6 +/- 0.6 log10 from baseline). Conversely the proportion of HIV-DNA carrying cells slightly declined at 3 months but remained substantially stable thereafter both in peripheral blood and in lymph nodes. Taken together these data suggest that this therapeutic regimen, although sub-optimal, is effective in significantly reducing the virus production by productively infected cells but does not seem to substantially affect the load of provirally infected cells.

CD4 Lymphocyte enumeration: comparison between flow cytometry and enzyme immunoassay.

Assessment of the CD4 lymphocyte number, currently performed by flow cytometry, is one of the main laboratory tests for establishing progression to acquired immunodeficiency syndrome (AIDS). An enzyme immunoassay has recently been commercialized which can be very useful for counting CD4 cells in laboratories where flow cytometers are not available. In the present study, a comparative evaluation of CD4 positive lymphocytes with flow cytometry and the enzyme assay was made in healthy subjects (N = 30, R = 0.88, P < 0.0001), human immunodeficiency virus (HIV)-infected individuals (N = 80, R = 0.91, P < 0.0001), and patients with autoimmune diseases (N = 28, R = 0.82, P < 0.001) or psoriasis (N = 18, R = 0.76, P = 0.01). A correlation between the two methodologies was not found in psoriatic patients under treatment with cyclosporin A (N = 7, R = 0.05, not significant). Some differences could be found at low CD4 lymphocyte levels since the influence of CD4 antigen eluted from monocytes or soluble CD4 in the whole blood sample could cause overestimation of CD4 cell numbers by the enzyme assay.

Serum IL-10 levels in HIV-positive subjects: correlation with CDC stages.

HIV-1 infection and the HIV gp120 have been shown to induce an IL-10 increase in cultured peripheral blood mononuclear cells. Furthermore, the expression of this cytokine has been reported to increase in lymphnodes of infected patients along the disease course, and a shift from the TH-1 towards the TH-0/TH-2 phenotypes (with subsequent IL-10 release) has been hypothesized to underly AIDS progression. In this study the serum IL-10 levels found in 30 HIV-negative controls and in 65 HIV-positive patients, untreated with AZT and negative for HBsAg and HCV-Ab have been compared, using a commercial, competitive ELISA method based on a polyclonal anti-IL-10 serum. With this test, HIV-positive sea showed IL-10 levels significantly higher than those found in the controls. In addition the IL-10 levels progressively increased in the subsequent CDC stages, without further changes from the stage III to the stage IV. Accordingly, patients evaluated two times in CDC stage II, with a time interval of at least one year, showed significant IL-10 increases, even more pronounced when the same patients passed from CDC stage II to stage III. Furthermore, a significant, negative correlation was observed between the circulating IL-10 levels and the patients' CD4/CD8 ratios. These data may be important from a clinical point of view since IL-10 monitoring could be considered as a surrogate marker for evaluating the disease progression. In addition, several immunological abnormalities present in HIV positive patients, such as the monocyte/macrophage impairment and the hypergammaglobulinemia could be related to the enhanced IL-10 expression.

Apoptosis in HIV infection: protective role of IL-2.

Peripheral blood mononuclear cells from HIV-infected subjects have been demonstrated by different methods to die by apoptosis after short time in culture. In the present study the percentages of apoptotic cells have been measured by propidium iodide staining and flow cytometry in PBMC from healthy controls (15) and HIV-infected subjects with asymptomatic (10) or advanced (15) disease, with or without anti-viral treatment. The percentage of apoptosis significantly correlated with clinical stage (CDCII: 15.85% +/- 9.17, CDCIV: 22.6% +/- 5.97, P < 0.001) and the CD4/CD8 CD3 cell ratio. R = -0.57, P = 0.012), while no differences were found in relation to AZT therapy. By adding IL-2 to the cultures the percentages of apoptosis of PBMC from HIV-infected patients were significantly reduced in all experiments.

Autoreactive response in seronegative homosexual men at high risk for HIV infection.

Peripheral autoreactive T cell response was evaluated by limiting dilution analysis of autologous mixed lymphocyte reaction cultures in 15 subjects at high risk for HIV infection and in 20 normal individuals. The two groups did not show a quantitative difference of peripheral autoreactive T cells, but they showed different kinetics. While controls provided a straight line passing through the origin, the majority of high risk individuals showed a curve with a limited linear portion at high cell concentration, indicating that different mechanisms regulate the autoreactive response in the two groups studied. A follow-up study performed in three high risk and three normal individuals revealed a time-dependent increase of peripheral autoreactive T cells only in high risk subjects. Such increase correlates with the decrease of CD4+ cell number and CD4+/CD8+ cell ratio. Furthermore, the proliferative response of the same three subjects to gp160 peptides suggests a specific cellular reactivity to HIV components. This work has potential importance in understanding some of the early events in HIV infection.

Serum reactivity against an immunoregulatory sequence of HIV p24 in HIV-1-infected subjects.

The aim of this study was to assess the antibody reactivity in HIV-infected subjects against an HIV-1 p24 sequence, p226 (aa226-237), including a seven amino acid epitope showing immunosuppressive activity in vitro and to evaluate the relationship between anti-peptide antibody levels and disease progression. Sera of HIV-infected subjects, at different stages of disease, were compared to control sera in a retrospective evaluation. Recombinant HIV-1 p24 and p24- and control-peptides were used in an enzyme immunoassay as targets for antibodies present in the sera. Antibodies directed against the whole p24 protein and its peptides were found in all the sera studied but at different levels. The anti-p226 reactivity was not significantly different at different clinical stages. Nevertheless, it was inversely correlated to the reactivity directed against the whole protein, that was lower in subjects characterized by low CD4 cell numbers.