The branched DNA hybridization assay has been improved by the inclusion of the novel nucleotides, isoC and isoG, in the amplification sequences to prevent non-specific hybridization. The novel isoC, isoG-containing amplification sequences have no detectable interaction with any natural DNA sequence. The control of non-specific hybridization in turn permits increased signal amplification. Addition of a 14 site preamplifier was found to increase the signal/noise ratio 8-fold. A set of 74 oligonucleotide probes was designed to the consensus HIV POL sequence. The detection limit of this new HIV branched DNA amplifier assay was approximately 50 molecules/ml. The assay was used to measure viral load in 87 plasma samples of HIV- infected patients on triple drug therapy whose RNA titers were <500 molecules/ml. In all 11 patients viral load eventually declined to below the detection limit with the new assay.

In studies monitoring disease progression and therapeutic response, it is essential that the method used for hepatitis C virus (HCV) quantification not be influenced by genotypic variability. The branched DNA assay provides a reliable method for the quantification of HCV RNA. A modified set of oligonucleotide probes for the branched DNA assay was developed to enhance the efficiency of binding to genotypic variants of HCV. The improved branched DNA assay (HCV RNA 2.0) yielded highly reproducible quantification of hepatitis C virus RNA and displayed a nearly 600-fold dynamic range in quantification up to 120 Meq of HCV RNA per ml. The quantification limit was set at 0.2 Meg of HCV RNA per ml to ensure a specificity of > or = 95%. With this lowered quantification limit and the enhanced hybridization of the probes, the HCV RNA 2.0 assay exhibited a high level of sensitivity (96%) and was virtually unaffected by the genotypic variability of HCV. The HCV RNA 2.0 assay may be a useful tool for following HCV RNA levels throughout the course of disease, selecting patients for therapy, and evaluating therapeutic response.

No abstract

Hepatitis G virus (HGV) is prevalent in patients with chronic liver disease and has been previously detected in liver specimens. However, it is unknown whether the virus is replicating in the liver or is simply a contaminant from serum. We sought to determine whether HGV was hepatotropic and to determine whether coinfection with HGV and hepatitis C virus (HCV) influenced the level of either virus. Virus was quantitated using branched DNA (bDNA) assay for both HGV and HCV in the liver explants and pre‐transplant serum samples from 30 transplant recipients: Group I, HGV/HCV coinfection (n  = 10); group II, HCV infection alone, (n = 8); group III, HGV alone (n = 12). In patients with coinfection HCV (RNA) titers in liver were consistently higher than those for HGV RNA (median 1.13 × 108 and 360,000 Eq/g respectively, P < .01). The ratio of liver/serum viral RNA was significantly higher for HCV than for HGV (median 129 and 0.3 respectively, P < .01). Levels of HCV RNA were similar in patients with HCV infection alone versus those with HGV/HCV coinfection (median; liver = 1.15 × 107 vs. 1.13 × 108 Eq/g, serum = 500,000 vs. 200,000 Eq/mL) and levels of HGV RNA in liver and serum were similar in patients with HGV infection alone compared to those with HGV/HCV coinfection (median; liver = 1.2 × 106 vs. 4.0 × 105 Eq/g, serum = 4.5 × 106 vs. 2.6 × 106 Eq/mL). Levels of either virus appeared unaffected by the presence of an additional virus. The high ratio of HCV RNA levels in liver compared to serum is consistent with its known hepatotropism, but this pattern was not observed for HGV. The median liver/serum ratio of HGV RNA was less than unity, a finding consistent with serum contamination of liver tissue. Thus we conclude that the liver is not the main site of HGV replication.

The quantification of human immunodeficiency virus type 1 (HIV-1) RNA has facilitated clinical research and expedited the development of antiretroviral drugs. The branched-DNA (bDNA) assay provides a reliable method for the quantification of HIV-1 RNA in human plasma and is considered one of the most reproducible assays ready for use in clinical trials. A series of oligonucleotide probe design and solution changes have been developed to enhance the sensitivity of the bDNA assay while maintaining its performance characteristics. Among the changes incorporated into the enhanced-sensitivity bDNA (ES bDNA) assay to reduce the background level and enhance the signal are the use of shorter overhang sequences of target probes for capture, the cruciform design of target probes for amplification, and the addition of preamplifier molecules. The ES bDNA assay is at least 20-fold more sensitive than the first-generation bDNA assay, yet it maintains a high level of accuracy, linearity, and reproducibility. Further, quantification values obtained with the ES bDNA assay and the first-generation bDNA assay are highly correlated, thus allowing for meaningful comparisons of HIV-1 RNA levels in specimens tested with either assay. The ES bDNA assay may be useful in determining the prognostic value of HIV-1 RNA levels of below 10,000 copies per ml and in assessing the clinical benefit of antiretroviral therapy-induced decreases in plasma HIV-1 RNA sustained at levels of below 10,000 copies per ml.