V. Sypsa

Development and assessment of a novel real-time PCR assay for quantitation of HBV DNA

HBV DNA quantitation is used extensively for the monitoring of treatment of hepatitis B virus (HBV) infection. The aim of this study was to develop a highly sensitive and reproducible real-time PCR (RTD-PCR) assay for the quantitation of HBV DNA using the LightCycler system. The performance of this assay was assessed by analyzing serial dilutions of HBV genomic DNA of known concentration and the lower limit of detection was found to be 1 DNA copy/reaction. By using serial dilutions of plasmid standard, RTD-PCR was determined to quantify HBV DNA in a 10-log10 dynamic range. RTD-PCR was found to be more sensitive than the commercially available tests such as the Quantiplex HBV DNA and the AMPLICOR HBV MONITOR assays. The median coefficient of variation of interexperimental variability was 3.2%. The HBV DNA values obtained with RTD-PCR were highly correlated with assays available commercially. These findings suggest that our RTD-PCR assay combines high sensitivity and reproducibility for HBV DNA quantitation in an incomparable high dynamic range of quantitation.

A viral kinetic study using pegylated interferon alfa‐2b and/or lamivudine in patients with chronic hepatitis B/HBeAg negative

We studied viral dynamic parameters in 44 chronic hepatitis B/hepatitis B e antigen (HBeAg)(−) patients treated with pegylated interferon alfa‐2b (PEG‐IFN) 100 or 200 μg weekly or lamivudine 100 mg daily or the combination of PEG‐IFN 100 or 200 μg with lamivudine. Patients receiving PEG‐IFN monotherapy exhibited viral load oscillations between weekly injections, which were resolved by the addition of lamivudine. The median pharmacological delay was estimated at 4.1, 5.8, and 1.8 hours in PEG‐IFN monotherapy, PEG‐IFN 100/200 μg + lamivudine, and lamivudine monotherapy, respectively (P = .44). The median half‐life of free virus was 12.7 hours (range, 2.4‐69.2 hours). The mean antiviral effectiveness of PEG‐IFN 100/200 μg monotherapy was lower than that of lamivudine (82.6% vs. 96.4%; P = .005). The mean effectiveness of PEG‐IFN 100 μg + lamivudine and PEG‐IFN 200 μg + lamivudine was 92.8% and 94.4%, respectively. The half‐life of infected cells ranged from 2.7 to 75 days. The median half‐life of infected cells in patients receiving the combination regimens of PEG‐IFN and lamivudine was similar to that of lamivudine patients (5.0 days vs. 6.0 days, P = .77). In conclusion, the addition of pegylated interferon alfa‐2b in lamivudine treatment was found to neither enhance the potency of blocking HBV production nor the decay rates of infected cells. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005;42:77–85.)

Quantitative Detection of the M204V Hepatitis B Virus Minor Variants by Amplification Refractory Mutation System Real-Time PCR Combined with Molecular Beacon Technology

ABSTRACT Mutations in the highly conserved tyrosine-methionine-aspartate-aspartate (YMDD) motif are frequently associated with resistance to antivirals and represent a major concern in the treatment of hepatitis B virus (HBV) infection. Conventional methods fail to detect minority populations of drug-resistant viral quasispecies if they represent less than 25% of the total sample virus population. The amplification refractory mutation system real-time PCR (ARMS RT-PCR) was combined with molecular beacon technology using the LightCycler system. The samples from HBV patients selected for assay evaluation included (i) 57 samples from treatment-naïve patients for biological discriminatory ability (cutoff) estimation, (ii) 12 samples from patients with treatment failure that were M204V positive by sequencing, and (iii) 13 samples from patients with treatment failure that were negative for mutation at codon 204 by sequencing. The discriminatory ability of the assay was 0.25% when tested with laboratory-synthesized DNA target sequences. The median mutant-to-wild-type ratio for samples from naive patients tested positive for the wild type and for mutant variants was 0.01% (5th and 95th percentiles = 0.0001 and 0.04%, respectively). A value of 0.04% was selected as the biological cutoff of the assay of clinical samples. In all samples M204V positive by sequencing (12/12), the mutant variant was detected as the predominant population (range, 82.76 to 99.43%). Interestingly, in 5 (38%) of 13 samples negative by sequencing, the M204V variant was detected at a ratio above the biological cutoff (0.05 to 28%). The assay represents an efficient technique for the early detection and quantification of M204V variants before mutant strains emerge to dominate the population.

Analytical and clinical sensitivity of the Procleix Ultrio HIV-1/HCV/HBV assay in samples with a low viral load

Background and Objectives  The Procleix Ultrio human immunodeficiency virus type 1 (HIV‐1)/hepatitis C virus (HCV)/hepatitis B virus (HBV) (Ultrio) assay simultaneously detects HIV‐1 RNA, HCV RNA and HBV DNA in individual blood donations. The main objective of the study was to assess the analytical and clinical sensitivity of the multiplex and discriminatory probe assays in samples with a low viral load.

Estimating the treatment cascade of chronic hepatitis B and C in Greece using a telephone survey

Accurate diagnosis and treatment rates for chronic hepatitis B (HBV) and C virus (HCV) infections are usually missing. Aim of this study was to estimate the HBV and HCV treatment cascade (proportion and absolute numbers of tested, aware/unaware, infected and treated) in Greek adults. A telephone survey was conducted in a sample representative of the Greek adult general population. Prevalence rates were age‐standardized for the Greek adult population and corrected for high‐risk individuals not included in the survey. Of the 9974 participants, 5255 (52.7%) had been tested for HBV and 2062 (20.7%) for HCV with the proportion varying according to age and being higher in middle‐age groups (P < 0.001). HBsAg was reported positive in 111/5255 (2.11%) and anti‐HCV in 26/2062 (1.26%) tested cases. The age‐adjusted prevalence was estimated to be 2.39% for HBV and 1.79% for HCV. Taking into account individuals at high risk for viral hepatitis not included in the survey, the ‘true’ prevalence was estimated to be 2.58% for HBV and 1.87% for HCV. Anti‐HBV and anti‐HCV treatment had been taken by 36/111 (32.4%) chronic HBV and 15/26 (57.7%) chronic HCV patients. In conclusion, almost 50% of chronic HBV and 80% of chronic HCV patients in Greece may be unaware of their infection, while only 32% or 58% of diagnosed chronic HBV or HCV patients, respectively, have been ever treated. Therefore, intensive efforts are required to improve the efficacy of screening for HBV and particularly for HCV as well as to reduce the barriers to treatment among diagnosed patients.

Genetic Evolution of Human Immunodeficiency Virus Type 1 in Two Spouses Responding Successfully to Highly Active Antiretroviral Therapy

The current case study provided an unusual setting to track the evolution of HIV-1 envelope gene over a maximum period of 6 years in two asymptomatic spouses undergoing suppressive highly active antiretroviral therapy. For this purpose, proviral DNA samples taken from uncultured peripheral blood mononuclear cells and spanning the C2-V5 regions of env were analyzed at three sampling points per subject. Two distinct topological patterns were observed in the phylogenetic reconstructions of the genetically linked sequences of the couple: an intermingled pattern and a sequentially shifting pattern in the virus populations of the male index case and his spouse, respectively. Application of three evolutionary models for the amino acid-encoded sites, using the maximum likelihood approach, indicated the operation of positive selection in the region only at the second time point in the woman, before receiving therapy. These findings reinforce the evidence of a crucial role for host-selective constraints on HIV-1 env evolution in vivo.

Erratum to “Development and assessment of a novel real-time PCR assay for quantitation of HBV DNA”: [J. Virol. Methods 103 (2002) 201–212]

D. Paraskevis , C. Haida , N. Tassopoulos , M. Raptopoulou , D. Tsantoulas , H. Papachristou , V. Sypsa , A. Hatzakis * a Department of Hygiene and Epidemiology, Athens University Medical School, M. Asias 75, GR-115 27, Athens, Greece b Western Attika General Hospital, Athens, Greece c Fourth Medical Department, University of Thessaloniki, Thessaloniki, Greece d ‘‘Sismanogleion’’ Hospital, Athens, Greece