Change In RNA Viral Load Research Articles

Longitudinal phenotypic analysis of human immunodeficiency virus type 1-specific cytotoxic T lymphocytes: correlation with disease progression.

Few studies have examined longitudinal changes in human immunodeficiency virus type 1 (HIV)-specific cytotoxic T lymphocytes (CTL). To more closely define the natural history of HIV-specific CTL, we used HLA-peptide tetrameric complexes to study the longitudinal CD8(+) T-cell response evolution in 16 A*0201-positive untreated individuals followed clinically for up to 14 years. As early as 1 to 2 years after seroconversion, we found a significant association between high frequencies of A*0201-restricted p17(Gag/Pol) tetramer-binding cells and slower disease progression (P < 0.01). We observed that responses could remain stable over many months, but any longitudinal changes that occurred were typically accompanied by reciprocal changes in RNA viral load. Phenotypic analysis with markers CD45RO, CD45RA, and CD27 identified distinct subsets of antigen-specific cells and the preferential loss of CD27(+) CD45RO(+) cells during periods of rapid decline in the frequency of tetramer-binding cells. In addition we were unable to confirm previous studies showing a consistent selective loss of HIV-specific cells in the context of sustained Epstein-Barr virus-specific cell frequencies. Overall, these data support a role of HIV-specific CTL in the control of disease progression and suggest that the ultimate loss of such CTL may be preferentially from the CD27(+) CD45RO(+) subset.

Use of multiple competitors for quantification of human immunodeficiency virus type 1 RNA in plasma.

Quantification of human immunodeficiency virus type 1 (HIV-1) RNA in plasma has rapidly become an important tool in basic HIV research and in the clinical care of infected individuals. Here, a quantitative HIV assay based on competitive reverse transcription-PCR with multiple competitors was developed. Four RNA competitors containing identical PCR primer binding sequences as the viral HIV-1 RNA target were constructed. One of the PCR primers was fluorescently labeled, which facilitated discrimination between the viral RNA and competitor amplicons by fragment analysis with conventional automated sequencers. The coamplification of known amounts of the RNA competitors provided the means to establish internal calibration curves for the individual reactions resulting in exclusion of tube-to-tube variations. Calibration curves were created from the peak areas, which were proportional to the starting amount of each competitor. The fluorescence detection format was expanded to provide a dynamic range of more than 5 log units. This quantitative assay allowed for reproducible analysis of samples containing as few as 40 viral copies of HIV-1 RNA per reaction. The within- and between-run coefficients of variation were <24% (range, 10 to 24) and <36% (range, 27 to 36), respectively. The high reproducibility (standard deviation, <0.13 log) of the overall procedure for quantification of HIV-1 RNA in plasma, including sample preparation, amplification, and detection variations, allowed reliable detection of a 0.5-log change in RNA viral load. The assay could be a useful tool for monitoring HIV-1 disease progression and antiviral treatment and can easily be adapted to the quantification of other pathogens.