The role of macrophages in the pathogenesis and progression of human immunodeficiency virus (HIV)-related infection is substantiated by in vitro and in vivo evidence. The unique ability to survive HIV infection and produce viral particles for long periods is postulated. Detailed studies of this phenomenon are lacking. The dynamics of HIV-1 replication and cumulative virus production was studied in long-term cultures of macrophages in the presence or in the absence of antiviral drugs. Multiply spliced and unspliced HIV-RNA production was assessed by quantitative PCR, and the number of infected cells was monitored by FACS analysis. Cumulative HIV-1 production was determined by a trapezoidal equation, including such parameters as times of collection and experimental values of genomic-RNA and p24 gag antigen. Unspliced and multiply spliced HIV-RNA increased linearly after macrophage infection; reached levels of 1.5 x 10(8) and 2.8 x 10(5) copies/10(5) cells, respectively, at day 10; and then remained stable throughout the course of the experiment. Cumulative production of genomic-RNA and p24 gag antigen was 10(10) copies/10(6) cells and 10(7) pg/10(6) cells, respectively, with an average of >200 virus particles produced daily by each macrophage. AZT decreased the cumulative production of both genomic-RNA and p24 gag antigen down to 2.5 x 10(9) copies and 1.1 x 10(6) pg/10(6) cells (73.8% and 88.9% inhibition, respectively) up to day 50 without virus breakthrough. Ritonavir had a limited, but consistent, efficacy on the release of mature virus proteins (about 40% inhibition), but not on HIV-RNA production. In conclusion, the long-term dynamics and the high cumulative virus production that characterize HIV-1 infection of macrophages underscore the peculiar role of these cells as a persistently infected reservoir of HIV.

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The new Abbott RealTime hepatitis B virus (HBV) assay was compared to the Cobas AmpliPrep/CobasTaqMan assay with 128 serum samples from patients with chronic hepatitis B. There was an excellent correlation (r ‫؍‬ 0.961) between the two assays, with the Abbott RealTime test showing at least equivalent sensitivity and a slightly wider dynamic range than the Cobas TaqMan assay. By coupling high sensitivity with a large dynamic range, the Abbott RealTime HBV assay is useful in monitoring the response to antiviral therapy.Hepatitis B virus (HBV) is a small partially double-stranded virus belonging to the family of the Hepadnaviridae that can cause acute or chronic hepatitis (2). Millions of people around the world are infected by HBV, and it is estimated that about 350 million people worldwide have developed a persistent infection (6). Measurement of HBV levels in serum is a reliable marker for prognosis of acute and chronic infection and plays an important role in the management of patients receiving antiviral drugs (7,8). Therefore, diagnostic assays for the accurate quantitation of HBV DNA are essential for optimal patient management. Monitoring of HBV DNA allows to predict the success of antiviral therapy and to identify the development of drug resistance. Real-time PCR is a new molecular tool that is progressively replacing endpoint PCR systems for monitoring patients with chronic hepatitis B since it allows for high sensitivity, a broad dynamic range, accuracy, and rapid results (1, 9).Quantification by real-time PCR is based on the determination of the threshold cycle (C T ) when the amplified product is detected for the first time (4) and the PCR is still in the exponential phase. In this case the quantification of the viral load is much more accurate and reliable than that measured with endpoint PCR systems (3).The aim of our study was to evaluate the Abbott RealTime HBV assay for the detection and quantification of HBV-DNA in serum samples and to compare these results with those obtained using the Cobas AmpliPrep/Cobas TaqMan HBV test (Roche Diagnostics). The study was carried out on 128 serum samples collected from patients with chronic HBV infections attending our hepatology clinic at the University Hospital Tor Vergata, Rome, Italy.Blood samples were collected in a BD Vacutainer tube containing a separation gel. After centrifugation, the serum was divided into two aliquots of 1 ml each and tested by the Abbott and Cobas TaqMan Real-time PCR assays, respectively. Both systems use a hybridization probe with a fluorescent moiety covalently linked to the 5Ј end of the probe (reporter) and a quenching moiety bound to the 3Ј end of the probe (quencher). In the presence of target, probe hybridization and primer extension occur simultaneously. The fluorescent signal is generated by removing the reporter through the 5Ј33Ј exonuclease activity of a thermostable Taq DNA polymerase (4, 5). In the TaqMan and Abbott RealTime PCR assays an additional probe is used to detect genotype G or C, respectively. While the Coba...

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Intrahepatic total HBV DNA (it-HBV DNA) level might reflect the size of virus reservoir and correlate with the histological status of the liver. To quantitate it-HBV DNA in a series of 70 liver biopsies obtained from hepatitis B chronic patients, a modified version of the COBASAmpliprep/COBASTaqMan HBV test v2.0 was used for this purpose. The linearity and reproducibility of the modified protocol was tested by quantifying serial dilutions of a full-length HBV containing plasmid and it-HBV DNA from a reference patient. A good linear trend between the expected values and those generated by the assay was observed at different concentrations of both plasmid and reference patient (R = 0.994 and 0.962, respectively). Differences between the values obtained in two independent runs were ≤0.3 log IU for the plasmid and ≤0.6 log IU/mg for the reference patient, showing a high inter-run reproducibility. In the 70 liver biopsies, it-HBV DNA level ranged from 1.4 to 5.4 log IU/mg, with a good linearity and reproducibility between the values obtained in two runs [R = 0.981; median (IQR) difference of it-HBV DNA 0.05 (0.02-0.09) IU/mg]. The modified COBASAmpliprep/COBASTaqMan HBV test v2.0 allows an accurate quantitation of it-HBV DNA. Its determination may have prognostic value and may be a useful tool for the new therapeutic strategies aimed at eradicating the HBV infection.

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